Neutralization of inhibitory pathways in lymphocytes

ABSTRACT

This invention relates to the use of NKG2A-neutralizing agents to treat cancers that are poorly responsive to PD-1 neutralizing agents, as well as to the combined use of NKG2A-neutralizing agents and PD-1 neutralizing agents for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/128,241, filed Dec. 21, 2020, which is a divisional of U.S.application Ser. No. 16/071,499, filed Jul. 20, 2018, now U.S. Pat. No.10,870,700, which is the U.S. national stage application ofInternational Patent Application No. PCT/EP2017/051153, filed Jan. 20,2017, which claims the benefit of U.S. Provisional Application No.62/281,217, filed Jan. 21, 2016, the disclosures of which areincorporated herein by reference in their entirety, including anydrawings.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“Seq-List.xml”, created Jun. 7, 2023, which is 21,085 bytes in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the use of NKG2A-neutralizing agents to treatcancers that are poorly responsive to PD-1 neutralizing agents, as wellas to the combined use of NKG2A-neutralizing agents and PD-1neutralizing agents for the treatment of cancer.

BACKGROUND OF THE INVENTION

NK cell activity is regulated by a complex mechanism that involves bothactivating and inhibitory signals. Several distinct NK-specificreceptors have been identified that play an important role in the NKcell mediated recognition and killing of HLA Class I deficient targetcells. Natural Cytotoxicity Receptors (NCR) refers to a class ofactivating receptor proteins, and the genes expressing them, that arespecifically expressed in NK cells. Examples of NCRs include NKp30,NKp44, and NKp46 (see, e.g., Lanier (2001) Nat Immunol 2:23-27, Pende etal. (1999) J Exp Med. 190:1505-1516, Cantoni et al. (1999) J Exp Med.189:787-796, Sivori et al. (1997) J. Exp. Med. 186:1129-1136, Pessino etal. (1998) J Exp Med. 188(5):953-60; Mandelboim et al. (2001) Nature409:1055-1060, the entire disclosures of which are herein incorporatedby reference). These receptors are members of the Ig superfamily, andtheir cross-linking, induced by specific mAbs, leads to a strong NK cellactivation resulting in increased intracellular Ca⁺⁺ levels, triggeringof cytotoxicity, and lymphokine release, and an activation of NKcytotoxicity against many types of target cells.

CD94/NKG2A is an inhibitory receptor found on subsets of lymphocytes.CD94/NKG2A restricts cytokine release and cytotoxic responses of certainlymphocytes towards cells expressing the CD94/NKG2A-ligand HLA-E (see,e.g., WO99/28748). HLA-E has also been found to be secreted in solubleform by certain tumor cells (Derre et al., J Immunol 2006; 177:3100-7)and activated endothelial cells (Coupe) et al., Blood 2007;109:2806-14). Antibodies that inhibit CD94/NKG2A signalling may increasethe cytokine release and cytolytic activity of lymphocytes towards HLA-Epositive target cells, such as responses of CD94/NKG2A-positive NK cellstowards HLA-E expressing tumor cells or virally infected cells.Therefore, therapeutic antibodies that inhibit CD94/NKG2A but that donot provoke the killing of CD94/NKG2A-expressing cells (i.e.non-depleting antibodies), may induce control of tumor-growth in cancerpatients.

PD-1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated Bcells, T cells, and myeloid cells Okazaki et al. (2002) Curr. Opin.Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8). Twoligands for PD-1 have been identified, PD-L1 and PD-L2, that have beenshown to downregulate T cell activation upon binding to PD-1 (Freeman etal. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). PD-L1 isabundant in a variety of human cancers (Dong et al. (2002) Nat. Med.8:787-9). The interaction between PD-1 and PD-L1 results in a decreasein tumor infiltrating lymphocytes, a decrease in T-cell receptormediated proliferation, and immune evasion by the cancerous cells.Immune suppression can be reversed by inhibiting the local interactionof PD-1 with PD-L1, and the effect is additive when the interaction ofPD-1 with PD-L2 is blocked as well.

PD-1 blockade has resulted in impressive anti-tumor responses innumerous clinical trials. However, in many cancers (e.g. lung cancer)not all patients respond to treatment with anti-tumor responses, andfurthermore some patients have cancers that relapse after treatment.Consequently, there is a need in the art for improved benefit topatients treated with inhibitors of the PD-1 axis.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides improved methods ofeliciting an anti-tumor immune response in an individual who is a poorresponder for treatment with a PD1-neutralizing agent (e.g. an anti-PD-1or anti-PD-L1 antibody), wherein the individual is treated with an agentthat neutralizes the inhibitory receptor NKG2A. Tumor-infiltratinglymphocytes in PD-1 poor responders can express the inhibitory receptorNKG2A following treatment with a PD-1 neutralizing antibody. In PD-1poor-responders, tumors may progress or escape immunosurveillancedespite treatment with a PD-1 neutralizing agent because TILs arerestricted by the inhibitory NKG2A receptor. A murine lymphoma model ofPD-1 resistance shows that treatment with an NKG2A-neutralizing agentinduces complete remission in the majority of individuals.

Accordingly, in one embodiment, provided is a method for treating orpreventing a cancer in an individual who is a poor responder or who hasa cancer that is poorly responsive (e.g., is observed to be a poorresponder or poorly responsive, or is predicted to be a poor responderor poorly responsive) to treatment with an agent that neutralizes theinhibitory activity of PD-1, the method comprising administering to theindividual a therapeutically active amount of a compound that inhibits ahuman NKG2A polypeptide. In one embodiment, a compound that inhibits ahuman NKG2A polypeptide is administered in combination with a compoundthat inhibits a human PD-1 polypeptide. In another embodiment, acompound that inhibits a human NKG2A polypeptide is administered withoutcombined treatment with a compound that inhibits a human PD-1polypeptide. In one embodiment, compound that inhibits a human NKG2Apolypeptide is administered during (in combination with) or followingthe end of (subsequent to) a course of treatment with a compound thatinhibits a human PD-1 polypeptide, optionally further wherein theindividual has experienced an incomplete response (or has notexperienced a complete response), cancer relapse or cancer progressionduring treatment with or following treatment (e.g. following a firstcourse or cycle of treatment) with a compound that inhibits a human PD-1polypeptide (e.g., in the absence of treatment with a compound thatinhibits a human NKG2A polypeptide), optionally wherein the individualhas (e.g. is determined to have) detectable and/or elevated numbers ofNKG2A-expressing NK and/or CD8 T cells and/or elevated levels of NKG2Aon NK and/or CD8 T cells. In one embodiment, the cancer is ahematological tumor, optionally a lymphoma or leukemia. In oneembodiment, the cancer is a carcinoma. In one embodiment, the individualhas detectable and/or elevated numbers of NKG2A-expressing NK and/or CD8T cells and/or elevated levels of NKG2A on NK and/or CD8 T cells.

In one embodiment, provided is a method for treating or preventing acancer in an individual comprising:

-   -   (a) administering to the individual an agent that neutralizes        the inhibitory activity of PD-1 (e.g. administering a cycle or        course of therapy with the agent); and    -   (b) if the cancer in the individual in step (a) is determined to        be poorly responsive to the agent that neutralizes the        inhibitory activity of PD-1 (e.g. is progressing, has not fully        responded or regressed, is non-responsive), administering to the        individual a therapeutically active amount of a compound that        inhibits a human NKG2A polypeptide (optionally in combination        with an agent that neutralizes the inhibitory activity of PD-1).

In one embodiment, the compound that inhibits a human NKG2A polypeptideis an antibody that neutralizes the inhibitory activity of NKG2A. In oneembodiment, the compound that inhibits a human PD-1 polypeptide is ananti-PD-1 or anti-PDL-1 antibody that neutralizes the inhibitoryactivity of PD-1. The individual can be specified to be a human.

In one embodiment, provided is a method of activating a CD8+tumor-infiltrating T cell in an individual who has a cancer that ispoorly responsive to treatment with an agent that neutralizes theinhibitory activity of PD-1 (e.g. is progressing, has not fullyresponded or regressed, is non-responsive), the method comprisingadministering to the individual a therapeutically active amount of acompound that inhibits a human NKG2A polypeptide.

In one aspect of any embodiment herein, an individual having a cancerthat is poorly responsive to treatment with an agent that neutralizesthe inhibitory activity of PD-1 is an individual who experiences or ispredicted to have a high likelihood to experience (e.g. based on one ormore prognostic factors) an incomplete response, lack of therapeuticresponse, detectable, relapsing or residual cancer and/or progressivedisease upon (during or following) treatment with an agent thatneutralizes the inhibitory activity of PD-1. In one embodiment, a cancerthat is poorly responsive is a cancer that has not responded, has notcompletely responded, remains detectable or residual, or has relapsed orprogressed despite (e.g. during or following) treatment with an agentthat neutralizes the inhibitory activity of PD-1, or which is predicted(e.g. based on one or more prognostic factors) to have a high likelihoodto not respond, to not completely respond, to remain detectable orresidual, or to relapsed or progress despite treatment with an agentthat neutralizes the inhibitory activity of PD-1. In one embodiment, anindividual who is a poor responder or who has a cancer that is poorlyresponsive is an individual who has experienced an incomplete response(e.g., has not experienced a complete response (CR)) upon (during orfollowing) treatment with an agent that neutralizes the inhibitoryactivity of PD-1. In one embodiment, an individual who is a poorresponder or who has a cancer that is poorly responsive is an individualwho has experienced at least a partial response (PR) upon (during orfollowing) treatment with an agent that neutralizes the inhibitoryactivity of PD-1, but whose cancer remains detectable, has relapsed orhas progressed.

In one embodiment, an individual who is a poor responder or who has acancer that is poorly responsive is an individual having a poor diseaseprognosis for treatment with an agent that neutralizes the inhibitoryactivity of PD-1 (e.g., in the absence of treatment with an agent thatneutralizes the inhibitory activity of NKG2A; as monotherapy). Anindividual having a poor disease prognosis can, for example, bedetermined to have a high or higher risk of cancer progression (e.g.compared to individuals having a good disease prognostic), based on oneor more predictive factors. In one embodiment, a predictive factor(s)comprises presence of detectable and/or elevated numbers ofNKG2A-expressing NK and/or CD8 T cells and/or elevated levels of NKG2Aon NK and/or CD8 T cells. In one embodiment, a predictive factor(s)comprises presence or absence of a mutation in one or more genes. In oneembodiment, the mutation defines a neo-epitope recognized by a T cell.In one embodiment, the predictive factor(s) comprises level(s) ofexpression of one or more genes or proteins in tumor cells, e.g. PD-L1,decreased or elevated levels of PD-L1 on tumor cells. In one embodiment,the predictive factor(s) comprises level(s) of expression of one or moregenes or proteins in NK and/or CD8 T cells in circulation or in thetumor environment, e.g., PD-1. In one embodiment, the predictivefactor(s) comprises mutational load in tumor cells, e.g. number ofnon-synonymous mutations per exome.

In one embodiment, an individual who is a poor responder or who has acancer that is poorly responsive is an individual having a cancer knownto be poorly responsive to treatment with a compound that inhibits ahuman PD-1 polypeptide, optionally a solid tumor, optionally ahaematological malignancy, optionally a head and neck squamous cellcarcinoma.

In one aspect provided is a composition comprising an antibody thatinhibits a human NKG2A polypeptide for use in the treatment orprevention of a cancer that is poorly responsive to treatment with acompound that inhibits a human PD-1 polypeptide (e.g. which hasprogressed or is predicted to progress following treatment with acompound that inhibits a human PD-1 polypeptide), optionally wherein thecancer is a solid carcinoma (e.g. a lung cancer), optionally wherein thecancer is a squamous cell carcinoma (e.g., a HNSCC), optionally whereinthe cancer is a haematological malignancy (e.g. a lymphoma).

In one aspect provided is agent that neutralizes the inhibitory activityof human NKG2A for use in combination with an agent that neutralizes theinhibitory activity of human PD-1, for treatment of cancers that arepoorly responsive to treatment with an agent that neutralizes theinhibitory activity of human PD-1. In one aspect provided is agent thatneutralizes the inhibitory activity of human NKG2A, in the treatment ofan individual having a lung cancer, a melanoma or a squamous cellcarcinoma (e.g., a HNSCC), the treatment comprising administering to theindividual an effective amount of each of: (a) an agent, optionally anantibody, that neutralizes the inhibitory activity of human NKG2A, and(b) an agent, optionally an antibody, that neutralizes the inhibitoryactivity of human PD-1.

In one aspect provided is a composition comprising an antibody thatinhibits a human NKG2A polypeptide for use in the treatment orprevention of a cancer in an individual who has received or isundergoing treatment with a compound that inhibits a human PD-1polypeptide, wherein the individual has detectable, increased and/orelevated numbers of NKG2A-expressing NK and/or CD8 T cells in the tumorenvironment and/or increased levels of expression of NKG2A on NK and/orCD8 T cells in the tumor environment. In one embodiment, the individualhas numbers of NK and/or CD8 T cells in circulation or in the tumorenvironment that are increased compared to numbers observed prior totreatment with a compound that inhibits a human PD-1 polypeptide, and/orthat are increased compared to a reference value (e.g. the number ofcells corresponds to values observed in patients who experience a poorresponse to a compound that inhibits a human PD-1 polypeptide). In oneembodiment, the individual has increased levels of expression of NKG2Aon NK and/or CD8 T cells in the tumor environment compared to levelsobserved prior to treatment with a compound that inhibits a human PD-1polypeptide, and/or compared to a reference value (e.g. the levelscorrespond to values observed in patients who experience poor responseto a compound that inhibits a human PD-1 polypeptide). In oneembodiment, the anti-NKG2A antibody is administered to an individual inan amount that results in the neutralization of the inhibitory activityof human CD94/NKG2A in a human patient (in vivo), e.g., an amount thatresults in the neutralization of the inhibitory activity of humanCD94/NKG2A on CD8 T cells and NK cells in a human patient. In oneembodiment, the amount that results in the neutralization of theinhibitory activity of human CD94/NKG2A in the human patient is at least10-fold (e.g., 10-20 fold, 10-50 fold, 10-100 fold, 20-50 fold, 20-100fold, 30-100 fold, 50-100 fold), optionally at least 50-, 60-, 80- or100-fold, the minimum concentration required to substantially saturateNKG2A receptors on the surface of NKG2A+ cells (e.g., in a binding assaywhere antibody is titrated on PBMC). In one embodiment, the anti-NKG2Aantibody competes with HLA-E for binding to human NKG2A.

In one aspect, it is shown herein that combined treatment with anti-NKGAantibodies and anti-PD-1 antibodies, or with anti-NKG2A antibodies andanti-PD-L1 antibodies, is particularly efficacious in treating cancers.It is also shown herein that treatment with anti-PD1 can causeupregulation of NKG2A receptors on tumor infiltrating lymphocytes, suchthat NKG2A may be restricting the efficacy of agents that block the PD1axis. Since these receptors can both restrict the cytotoxic activitiesof tumor infiltrating lymphocytes, neutralization of the inhibitoryactivity of both these two receptors by antibodies enables NKG2A+PD1+lymphocytes to effectively eliminate cancer cells. In one embodiment,the NKG2A+PD1+ lymphocytes are cytotoxic lymphocytes, optionally CD8+ Tcells or NK cells.

In one aspect, the present invention provides improved methods ofenhancing an anti-tumor immune response through the combinedneutralization of inhibitory receptors NKG2A and PD-1, e.g. via the useof antibodies. The combined treatment can be used to treat an individualhaving a cancer (e.g. a cancer known to be poorly responsive to agentthat neutralize PD-1, a non-small cell lung cancer (NSCLC), kidneycancer, gastrointestinal cancer, pancreatic or esophagus adenocarcinoma,breast cancer, renal cell carcinoma (RCC), melanoma, colorectal canceror ovarian cancer), irrespective of whether they are poor responders toneutralization of the inhibitory activity of PD-1, and thus including,e.g., individuals that are poor responders to treatment with an agentthat neutralizes the inhibitory activity of PD-1 and individuals thatare good responders to treatment with an agent that neutralizes theinhibitory activity of PD-1.

Accordingly, in one embodiment, provided is a method for treating orpreventing a cancer which is poorly responsive to treatment with acompound that inhibits a human PD-1 polypeptide, the method comprisingadministering to the individual having the cancer: (a) a therapeuticallyactive amount of a compound that inhibits a human NKG2A polypeptide, and(b) a therapeutically active amount of a compound that inhibits a humanPD-1 polypeptide. In one embodiment, the cancer is a solid tumor,optionally a solid tumor comprising infiltrating NK and/or CD8 T cells,optionally wherein the NK and/or CD8 T cells express NKG2A, optionallywherein at least 10%, 20%, 30%, 40% or 50% of the NK cells express NKG2Aat their surface. In one embodiment, the compound that inhibits a humanNKG2A polypeptide is an antibody that neutralizes the inhibitoryactivity of NKG2A. In one embodiment, the compound that inhibits a humanPD-1 polypeptide is an anti-PD-1 or anti-PDL-1 antibody that neutralizesthe inhibitory activity of PD-1. The individual can be specified to be ahuman.

In one embodiment, provided is method of activating or potentiating theactivity of a CD8+ tumor-infiltrating T cell in an individual, themethod comprising administering to an individual: (a) a therapeuticallyactive amount of a compound that inhibits a human NKG2A polypeptide, and(b) a therapeutically active amount of a compound that inhibits a humanPD-1 polypeptide. In one embodiment, provided is method of activating orpotentiating the activity of a tumor-infiltrating NK cell in anindividual, the method comprising administering to an individual: (a) atherapeutically active amount of a compound that inhibits a human NKG2Apolypeptide, and (b) a therapeutically active amount of a compound thatinhibits a human PD-1 polypeptide. In one embodiment, the cancer is asolid tumor. Optionally, in any embodiment, the individual has a cancerpoorly responsive to treatment with a compound that inhibits a humanPD-1 polypeptide.

In one aspect, the invention provides a treatment comprisingadministering a combination of an antibody that neutralizes theinhibitory activity of NKG2A, and antibody that neutralizes theinhibitory activity of PD-1. In one aspect of any embodiment herein,provided is a composition comprising an antibody that inhibits a humanNKG2A polypeptide and an antibody that binds PDL1 or PD1 and neutralizesthe inhibitory activity of the human PD-1 polypeptide. In one aspect,the composition is used in the treatment or prevention of a cancer.

In one aspect, an antibody that neutralizes the inhibitory activity ofNKG2A and antibody that neutralizes the inhibitory activity of PD-1 areused in combination in the treatment or prevention of a cancer known tobe poorly responsive to treatment with a compound that inhibits a humanPD-1 polypeptide, optionally a solid tumor, optionally a haematologicalmalignancy. In one embodiment, the cancer known to be poorly responsiveis a squamous cell carcinoma, optionally a head and neck squamous cellcarcinoma.

In any embodiment herein, the compound or agent that inhibits a humanPD-1 polypeptide comprises a polypeptide (e.g. an antibody, apolypeptide fused to an Fc domain, an immunoadhesin, etc.) that preventsPD-L1-induced PD-1 signalling, e.g. by blocking the interaction betweenPD-1 and its natural ligand PD-L1 (and optionally further blocking theinteraction between PD-1 and PD-L2. In one aspect the polypeptide is anantibody that binds PD-1 (an anti-PD-1 antibody); such antibody mayblock the interaction between PD-1 and PD-L1 and/or between PD-1 andPD-L2. In another aspect the polypeptide is an antibody that binds PD-L1(an anti-PD-L1 antibody) and blocks the interaction between PD-1 andPD-L1. In one embodiment, the antibody that neutralizes a human PD-1polypeptide is/has been administered in an amount that results in theneutralization of the inhibitory activity of human PD-1 in the humanpatient (in vivo), e.g. an amount that results in the neutralization ofthe inhibitory activity of human PD-1 on CD8 T cells and NK cells in thehuman patient. In one aspect, the antibody is administered (or is foradministration) according to a particular clinical dosage regimen,notably at a particular dose amount and according to a specific dosingschedule.

In one aspect of any embodiment herein, the compound or agent thatneutralizes the inhibitory receptor NKG2A is an antibody. In one aspect,an antibody that neutralizes NKG2A is a non-depleting antibody, e.g. anantibody that does not kill, eliminate, lyse or induce such killing,elimination or lysis, so as to negatively affect the number ofNKG2A-expressing cells present in a sample or in a subject. In oneaspect an antibody that neutralizes NKG2A is a non-depleting antibody. Anon-depleting antibody can, for example, lack an Fc domain or have an Fcdomain with minimal or no binding to one or more Fcγ receptors (e.g.CD16). Example include antibodies with constant regions from human IgG4isotype antibodies, antibodies of any isotype (e.g. IgG1, IgG2, IgG3,IgG4) with constant regions modified to reduce or abolish binding to oneor more Fcγ receptors (e.g. CD16, CD32A, CD32B and/or CD64).

In one aspect of any embodiment herein, the anti-NKG2A antibody isadministered for at least one administration cycle, the administrationcycle comprising at least a first and second (and optionally a 3^(rd),4^(th), 5^(th), 6^(th), 7^(th) and/or 8^(th) or further) administrationof the anti-NKG2A antibody, wherein the anti-NKG2A antibody isadministered in an amount effective to achieve a continuous (minimum)blood concentration of anti-NKG2A antibody of at least 10 μg/ml (or,optionally at least 20, 30, 40 or 50 μg/mL) between the first and second(and optionally the further) administrations. Achieving or maintaining aspecified continuous blood concentration means that the bloodconcentration does not drop substantially below the specified bloodconcentration for the duration of the specified time period (e.g.between two administrations of antibody, number of weeks), i.e. althoughthe blood concentration can vary during the specified time period, thespecified blood concentration represents a minimum or “trough”concentration.

In one embodiment, the anti-NKG2A antibody is administered in an amounteffective to achieve a peak blood concentration of about or at leastabout 50, 60, 70 or 80 μg/ml, optionally at least about 100 μg/ml, uponadministration (e.g. within 1 or 2 days of administration).

In one embodiment, the anti-NKG2A antibody is administered in an amounteffective to achieve a continuous (minimum) blood concentration ofanti-NKG2A antibody of about or at least about 10, 20, 30, 40, 50, 60,70 or 80 μg/ml, optionally at least about 100 μg/ml, for at least oneweek, or at least two weeks, following administration of the antibody.

In one embodiment, the anti-NKG2A antibody is administered in an amounteffective to achieve a continuous (minimum) blood concentration ofanti-NKG2A antibody of about or at least about 50, 60, 70 or 80 μg/ml,optionally at least about 100 μg/ml, between two successiveadministrations. In one embodiment, the first and second administrationsare separated in time by about two weeks, optionally about one week.

The anti-NKG2A antibody can optionally be administered in an amounteffective and according to a frequency that achieves a continuous(minimum) blood concentration as specified for the entire duration of anadministration cycle.

In one embodiment, the antibody that inhibits a human NKG2A polypeptideis administered subsequently to a course of treatment (the course oftreatment may be fully completed or stopped prior to completion, orsubsequently to the initiation of a course of treatment (e.g., in caseof an incomplete response, tumor progression, etc.) with an antibodythat neutralizes a human PD-1 polypeptide, in an administration cyclecomprising least two administrations of the anti-NKG2A antibody. Inanother embodiment, the antibody that inhibits a human NKG2A polypeptideis administered in an administration cycle in combination with antibodythat neutralizes a human PD-1 polypeptide, wherein the administrationcycle comprises least two administrations of the anti-NKG2A antibody. Inone embodiment, the anti-NKG2A antibody is administered in an amounteffective to achieve a continuous (minimum) concentration in anextravascular tissue (e.g. in the tumor environment) of at least 4μg/mL, optionally at least 10 μg/mL between two successiveadministrations. Optionally, the anti-NKG2A antibody is administered inan amount effective to achieve a continuous (minimum) concentration inan extravascular tissue (e.g. in the tumor environment) of at least 4μg/mL, optionally at least 10 μg/mL, for the entire duration of theadministration cycle. In one embodiment, the anti-NKG2A antibody isadministered in an amount effective to achieve a continuous (minimum)blood concentration of anti-NKG2A antibody of at least 40 μg/mL,optionally at least 100 μg/mL, between two successive administrations,or for the duration of the administration cycle.

In one embodiment the cancer is hematological cancer. In onenon-limiting embodiment, the cancer is a lymphoma or a leukemia. In oneembodiment the cancer is an advanced and/or refractory solid tumor. Inone non-limiting embodiment, the cancer (e.g., the advanced refractorysolid tumor) is selected from the group consisting of non-small celllung cancer (NSCLC), kidney cancer, gastrointestinal cancer, pancreaticor esophagus adenocarcinoma, breast cancer, renal cell carcinoma (RCC),melanoma, colorectal cancer, and ovarian cancer.

The compound that inhibits a NKG2A polypeptide (anti-NKG2A agent) is acompound that increases the ability of an NKG2A-expressing NK and/or Tcells to cause the death of the HLA-E-expressing cell. Optionally, thecompound that inhibits a NKG2A polypeptide is a polypeptide, optionallyan antibody (e.g. monoclonal antibody), that binds a NKG2A polypeptide.

In one embodiment, the anti-NKG2A agent reduces the inhibitory activityof NKG2A by blocking binding of its ligand, HLA-E, i.e., the anti-NKG2Aagent interferes with the binding of NKG2A by HLA-E. Antibody having theheavy chain of any of SEQ ID NOS: 4-8 and the light chain of SEQ ID NO:9 is an example of such an antibody. In one embodiment, the anti-NKG2Aagent reduces the inhibitory activity of NKG2A without blocking bindingof its ligand, HLA-E, i.e., the anti-NKG2A agent is a non-competitiveantagonist and does not interfere with the binding of NKG2A by HLA-E.The antibody having the heavy and light chain variable regions of SEQ IDNOS: 10 and 11 respectively is an example of such an antibody.

In one embodiment, the anti-NKG2A agent is antibody which binds with asignificantly higher affinity to NKG2A than to one or more activatingNKG2 receptors. For example, in one embodiment, the agent is antibodywhich binds with a significantly higher affinity to NKG2A than to NKG2C.In an additional or alternative embodiment, the agent is antibody whichbinds with a significantly higher affinity to NKG2A than to NKG2E. In anadditional or alternative embodiment, the agent is an antibody whichbinds with a significantly higher affinity to NKG2A than to NKG2H.

In one embodiment, the anti-NKG2A agent competes with the antibodyhaving the heavy and light chains of SEQ ID NOS: 4-8 and 9 respectively,or the antibody having the heavy and light chain variable regions of SEQID NOS: 10 and 11 respectively, in binding to CD94/NKG2A. The agent canbe, e.g., a human or humanized anti-NKG2A antibody.

In one embodiment, the anti-NKG2A antibody is a humanized antibodyhaving the heavy chain CDRs of any of the heavy chains of any of SEQ IDNOS: 4-8 and the light chain CDRs of the light chain of SEQ ID NO: 9respectively. In one embodiment, the anti-NKG2A antibody is a humanizedantibody having the heavy chain variable region of any of the heavychains of any of SEQ ID NOS: 4-8 and the light chain variable region ofthe light chain of SEQ ID NO: 9 respectively. Exemplarycomplementarity-determining region (CDR) residues or sequences and/orsites for amino acid substitutions in framework region (FR) of suchhumanized antibodies having improved properties such as, e.g., lowerimmunogenicity, improved antigen-binding or other functional properties,and/or improved physicochemical properties such as, e.g., betterstability, are provided.

In certain optional aspects, provided is a method for identifying anindividual having a cancer who is a poor responder to treatment with anagent that neutralizes the inhibitory activity of human PD-1, the methodcomprising:

a) determining levels of NKG2A (and optionally further PD-1) expressionon NK and/or CD8 T cells and/or numbers of NKG2A⁺ (optionallyNKG2A⁺PD1⁺) NK and/or CD8 T cells in an individual who has been treatedwith an agent that neutralizes the inhibitory activity of human PD-1(e.g. who has received at least one administration of the agent); and

b) upon a determination that the individual has increased levels ofNKG2A expression on NK and/or CD8 T cells (optionally on PD-1-expressingNK or T cells) and/or increased numbers of NKG2A⁺ (optionallyNKG2A⁺PD1⁺) NK and/or CD8 T cells (e.g. compared to a reference value,increased compared to reference value, increased compared to a valueobserved prior to treatment with the agent), identifying the individualas a poor responder to treatment with an agent that neutralizes theinhibitory activity of human PD-1.

In certain optional aspects, patients can be identified for treatmentwith an anti-NKG2A agent by assessing the presence in a tumor sample(e.g. tumor tissue and/or tumor adjacent tissue) of NKG2A expression onNK and/or CD8+ T cells. In one embodiment of any of the therapeutic usesor cancer treatment or prevention methods herein, the treatment orprevention of a cancer in an individual comprises:

a) determining levels of NKG2A expression on NK and/or CD8 T cellsand/or numbers of NKG2A-expressing NK and/or CD8 T cells in anindividual having a cancer who has been or who is being treated with anagent that neutralizes the inhibitory activity of PD-1, and

b) upon a determination that the individual has increased levels ofNKG2A expression on NK and/or CD8 T cells and/or increased numbers ofNKG2A-expressing NK and/or CD8 T cells, administering to the individuala compound that neutralizes the inhibitory activity of a human NKG2Apolypeptide.

In one embodiment of any of the methods, determining levels of NKG2Aexpression on and/or numbers of NKG2A+ NK and/or CD8 T cells comprisesdetermining the level of expression of a NKG2A nucleic acid orpolypeptide on NK and/or CD8 T cells and/or determining the number ofNKG2A+ NK and/or CD8 T cells in a biological sample and comparing thelevel to a reference level (e.g. a value, weak or strong cell surfacestaining, etc.). The reference level may, for example, correspond to ahealthy individual, to an individual responsive or poorly-responsive totreatment with an agent that inhibits a human PD-1 polypeptide, to anindividual deriving no/low clinical benefit from treatment with ananti-NKG2A antibody, or to an individual deriving substantial clinicalbenefit from treatment with an anti-NKG2A antibody (optionally incombination with an agent that inhibits a human PD-1 polypeptide). Adetermination that a biological sample comprises numbers ofNKG2A-expressing NK and/or CD8 T cells that are increased (e.g. at anumber that corresponds to that of an individual not deriving sufficientclinical benefit from treatment with an agent that inhibits a human PD-1polypeptide, a number that corresponds to that of an individual derivingsubstantial clinical benefit from treatment with an anti-NKG2A antibody)indicates that the individual has a cancer that can be treated with ananti-NKG2A antibody, e.g. according to the treatment methods describedherein. A determination that a biological sample expresses NKG2A nucleicacid or polypeptide at a level that is increased (e.g. a high value,strong surface staining, a level that corresponds to that of anindividual not deriving sufficient clinical benefit from treatment withan agent that inhibits a human PD-1 polypeptide, a level thatcorresponds to that of an individual deriving substantial clinicalbenefit from treatment with an anti-NKG2A antibody, a level that ishigher than that corresponding to an individual deriving no/low clinicalbenefit from treatment with an anti-NKG2A antibody, etc.) indicates thatthe individual has a cancer that can be treated with an anti-NKG2Aantibody, e.g. according to the treatment methods described herein.

In one embodiment, the CD8 T cells are tumor infiltrating CD8 T cells.In one embodiment, the NK cells are tumor infiltrating NK cells. In oneembodiment, at least 15%, 20 25% or 30% of NK and/or CD8 T cells arePD1⁺NKG2A⁺.

In one embodiment, the individual has a cancer comprising malignantcells that express a HLA-E polypeptide at their surface. In oneembodiment, a method of treatment further comprises determining theHLA-E polypeptide status of malignant cells (e.g. tumor cells) withinthe individual having a cancer, wherein a determination that malignantcells express HLA-E nucleic acid or polypeptide indicates that theindividual has a cancer that can be treated with an agent that inhibitsNKG2A.

In other embodiments, pharmaceutical compositions and kits are provided,as well as methods for using them. In one embodiment, provided is apharmaceutical composition comprising a compound that neutralizes theinhibitory activity of a human NKG2A polypeptide. In one embodiment,provided is a kit comprising a compound that neutralizes the inhibitoryactivity of a human NKG2A polypeptide and an agent capable of detectingthe expression of NKG2A on the surface of NK and/or CD8 T cells (e.g. anantibody or other NKG2A-binding agent bound to a detectable moiety).

These aspects are more fully described in, and additional aspects,features, and advantages will be apparent from, the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show PD-L1+Qa-1+ RMA-S Qa-1 Qdm B2m and A20 tumor cellsare infiltrated by NK cells expressing NKG2A and CD8 T cells expressingNKG2A and/or PD-1.RMA-S Qa-1 Qdm B2m (top row) and A20 (bottom row)tumor bearing mice were sacrificed when tumor volumes were around 500mm³. Tumor cells (FIG. 1A) and tumor infiltrating lymphocytes-TIL- (FIG.1B) were analyzed by flow cytometry respectively for the expression ofQa-1 and PDL-1 for tumor cells and NKG2A/C/E and PD-1 for TIL.MFI:Median of fluorescence intensity.

FIG. 2 shows distribution of NKG2A and PD-1 on NK and T cell subsets inmice. Lymphocytes were taken from spleen, from tumor draining lymphnodes, and from within solid tumor masses. PD-1 expression wasinfrequent or absent among all cell subsets from spleen and lymph nodes,however among tumor infiltrating lymphocytes (TIL), all cells subsetshad relatively high percentages of cells expressing PD-1. NKG2A on theother hand was found on NK cells but not on T cell subsets in spleen andlymph nodes, yet in the tumor was found on a significant percentage ofthe TILs, with a mean of more than 30% of NK cells and more than 19% ofCD8 T cells double positive for NKG2A and PD-1.

FIGS. 3A and 3B show NKG2A and PD-1 expression in tumor bearing mice.RMA Rae1 (top row), MC38 (medium row) and RMA (bottom row) tumor bearingmice were sacrificed when their tumors reached respectively the volumesof 500, 2000 and 800 mm³. NK cells (FIG. 3A) and CD8 T cells (FIG. 3B)were analyzed by flow cytometry in spleen, tumor draining lymph node(LN) and tumor for NKG2A/C/E and PD-1 expression.

FIG. 4 shows treatment of mice with anti-PD-1 mAb increases thefrequency of NKG2A expressing TCD8 cells in MC38 tumors. MC38 tumorbearing mice were either treated with 200 μg of rat IgG2a isotypecontrol (IC) or anti-mouse PD-1 antibodies on days 11, 14 and 17 aftercells engraftment. Mice were sacrificed on day 31 and CD8 T cells werecharacterized by flow cytometry in spleen, tumor draining lymph node(LN) and tumor.

FIG. 5 shows median tumor volume over time in mice treated with isotypecontrol, anti-mouse NKG2A mAb (200 μg, iv), anti-mouse PD-L1 mAb (200μg, ip) or anti-mNKG2A/mPDL-1 combination on days 11, 14 and 18. Whileanti-NKG2A yielded only a modest anti-tumor effect compared to isotypecontrol in this model and anti-PD-L1 yielded a substantial anti-tumoreffect but with tumor volume increasing toward day 28, the combinedtreatment with anti-NKG2A and anti-PD-L1 completely abolished tumorgrowth, with no significant growth in tumor volume observed at day 28.

FIG. 6 shows median tumor volume over time in mice bearing PD-1/-L1resistant A20 lymphomas treated with isotype control or neutralizinganti-mouse PD-L1 mAb or anti-mouse PD-1. Neither isotype control,anti-PD1 or anti-PD-L1 antibodies were effective in controlling tumorgrowth.

FIG. 7 shows median tumor volume over time in mice bearing A20 lymphomastreated with isotype control or neutralizing anti-mouse PD-1 mAb oranti-mouse PD-1 in combination with neutralizing anti-mouse NKG2A.Neither isotype control or anti-PD1 antibodies were effective incontrolling tumor growth, however when anti-NKG2A antibody treatment wasadded, complete tumor regression was observed in the majority of animals(7/10).

FIG. 8 shows median tumor volume over time in mice bearing A20 lymphomastreated with isotype control or neutralizing anti-mouse PD-L1 mAb oranti-mouse PD-L1 in combination with neutralizing anti-mouse NKG2A. Whenanti-NKG2A antibody treatment was added to anti-PD-L1 treatment,complete tumor regression was observed in the majority of animals(9/11).

DETAILED DESCRIPTION Definitions

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

Where “comprising” is used, this can optionally be replaced by“consisting essentially of” or by “consisting of”.

NKG2A (OMIM 161555, the entire disclosure of which is hereinincorporated by reference) is a member of the NKG2 group of transcripts(Houchins, et al. (1991) J. Exp. Med. 173:1017-1020). NKG2A is encodedby 7 exons spanning 25 kb, showing some differential splicing. Togetherwith CD94, NKG2A forms the heterodimeric inhibitory receptor CD94/NKG2A,found on the surface of subsets of NK cells, α/β T cells, γ/δ T cells,and NKT cells. Similar to inhibitory KIR receptors, it possesses an ITIMin its cytoplasmic domain. As used herein, “NKG2A” refers to anyvariant, derivative, or isoform of the NKG2A gene or encoded protein.Human NKG2A comprises 233 amino acids in 3 domains, with a cytoplasmicdomain comprising residues 1-70, a transmembrane region comprisingresidues 71-93, and an extracellular region comprising residues 94-233,of the following sequence:

(SEQ ID NO: 1) MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL.

NKG2C (OMIM 602891, the entire disclosure of which is hereinincorporated by reference) and NKG2E (OMIM 602892, the entire disclosureof which is herein incorporated by reference) are two other members ofthe NKG2 group of transcripts (Gilenke, et al. (1998) Immunogenetics48:163-173). The CD94/NKG2C and CD94/NKG2E receptors are activatingreceptors found on the surface of subsets of lymphocytes such as NKcells and T-cells.

HLA-E (OMIM 143010, the entire disclosure of which is hereinincorporated by reference) is a nonclassical MHC molecule that isexpressed on the cell surface and regulated by the binding of peptides,e.g. such as fragments derived from the signal sequence of other MHCclass I molecules. Soluble versions of HLA-E have also been identified.In addition to its T-cell receptor binding properties, HLA-E bindssubsets of natural killer (NK) cells, natural killer T-cells (NKT) and Tcells (α/β and y/O), by binding specifically to CD94/NKG2A, CD94/NKG2B,and CD94/NKG2C (see, e.g., Braud et al. (1998) Nature 391:795-799, theentire disclosure of which is herein incorporated by reference). Surfaceexpression of HLA-E protects target cells from lysis by CD94/NKG2A+NK,T, or NKT cell clones. As used herein, “HLA-E” refers to any variant,derivative, or isoform of the HLA-E gene or encoded protein.

In the context of the present invention, “NKG2A” or “CD94/NKG2A positivelymphocyte” refers to cells of the lymphoid lineage (e.g. NK-, NKT- andT-cells) expressing CD94/NKG2A on the cell-surface, which can bedetected by e.g. flow-cytometry using antibodies that specificallyrecognize a combined epitope on CD94 and NKG2A or and epitope on NKG2Aalone. “NKG2A positive lymphocyte” also includes immortal cell lines oflymphoid origin (e.g. NKL, NK-92).

In the context of the present invention, “reduces the inhibitoryactivity of NKG2A”, “neutralizes NKG2A” or “neutralizes the inhibitoryactivity of NKG2A” refers to a process in which CD94/NKG2A is inhibitedin its capacity to negatively affect intracellular processes leading tolymphocyte responses such as cytokine release and cytotoxic responses.This can be measured for example in a NK- or T-cell based cytotoxicityassay, in which the capacity of a therapeutic compound to stimulatekilling of HLA-E positive cells by CD94/NKG2A positive lymphocytes ismeasured. In one embodiment, an antibody preparation causes at least a10% augmentation in the cytotoxicity of a CD94/NKG2A-restrictedlymphocyte, optionally at least a 40% or 50% augmentation in lymphocytecytotoxicity, optionally at least a 70% augmentation in NKcytotoxicity”, and referring to the cytotoxicity assays described. If ananti-NKG2A antibody reduces or blocks CD94/NKG2A interactions withHLA-E, it may increase the cytotoxicity of CD94/NKG2A-restrictedlymphocytes. This can be evaluated, for example, in a standard 4-hour invitro cytotoxicity assay using, e.g., NK cells that express CD94/NKG2A,and target cells that express HLA-E. Such NK cells do not efficientlykill targets that express HLA-E because CD94/NKG2A recognizes HLA-E,leading to initiation and propagation of inhibitory signaling thatprevents lymphocyte-mediated cytolysis. Such an in vitro cytotoxicityassay can be carried out by standard methods that are well known in theart, as described for example in Coligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993). Chromium release and/or other parameters to assess theability of the antibody to stimulate lymphocytes to kill target cellssuch as P815, K562 cells, or appropriate tumor cells are also disclosedin Sivori et al., J. Exp. Med. 1997; 186:1129-1136; Vitale et al., J.Exp. Med. 1998; 187:2065-2072; Pessino et al. J. Exp. Med. 1998;188:953-960; Neri et al. Clin. Diag. Lab. Immun. 2001; 8:1131-1135;Pende et al. J. Exp. Med. 1999; 190:1505-1516, the entire disclosures ofeach of which are herein incorporated by reference. The target cells arelabeled with 51Cr prior to addition of NK cells, and then the killing isestimated as proportional to the release of 51Cr from the cells to themedium, as a result of killing. The addition of an antibody thatprevents CD94/NKG2A from binding to HLA-E results in prevention of theinitiation and propagation of inhibitory signaling via CD94/NKG2A.Therefore, addition of such agents results in increases inlymphocyte-mediated killing of the target cells. This step therebyidentifies agents that prevent CD94/NKG2A-induced negative signaling by,e.g., blocking ligand binding. In a particular 51Cr-release cytotoxicityassay, CD94/NKG2A-expressing NK effector-cells can kill HLA-E-negativeLCL 721.221 target cells, but less well HLA-E-expressing LCL 721.221-Cw3control cells. In contrast, YTS effector-cells that lack CD94/NKG2A killboth cell-lines efficiently. Thus, NK effector cells kill lessefficiently HLA-E⁺ LCL 721.221-Cw3 cells due to HLA-E-induced inhibitorysignaling via CD94/NKG2A. When NK cells are pre-incubated with blockinganti-CD94/NKG2A antibodies according to the present invention in such a51Cr-release cytotoxicity assay, HLA-E-expressing LCL 721.221-Cw3 cellsare more efficiently killed, in an antibody-concentration-dependentfashion. The inhibitory activity (i.e. cytotoxicity enhancing potential)of an anti-NKG2A antibody can also be assessed in any of a number ofother ways, e.g., by its effect on intracellular free calcium asdescribed, e.g., in Sivori et al., J. Exp. Med. 1997; 186:1129-1136, thedisclosure of which is herein incorporated by reference. Activation ofNK cell cytotoxicity can be assessed for example by measuring anincrease in cytokine production (e.g. IFN-γ production) or cytotoxicitymarkers (e.g. CD107 or CD137 mobilization). In an exemplary protocol,IFN-γ production from PBMC is assessed by cell surface andintracytoplasmic staining and analysis by flow cytometry after 4 days inculture. Briefly, Brefeldin A (Sigma Aldrich) is added at a finalconcentration of 5 μg/ml for the last 4 hours of culture. The cells arethen incubated with anti-CD3 and anti-CD56 mAb prior to permeabilization(IntraPrep™; Beckman Coulter) and staining with PE-anti-IFN-γ or PE-IgG1(Pharmingen). GM-CSF and IFN-γ production from polyclonal activated NKcells are measured in supernatants using ELISA (GM-CSF: DuoSet Elisa,R&D Systems, Minneapolis, MN, IFN-γ: OptEIA set, Pharmingen).

As used herein, the terms “PD-1” refers to the protein Programmed Death1 (PD-1) (also referred to as “Programmed Cell Death 1”), an inhibitorymember of the CD28 family of receptors, that also includes CD28, CTLA-4,ICOS and BTLA. The complete human PD-1 sequence can be found underGenBank Accession No. U64863, shown as follows:

(SEQ ID NO: 2) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFFPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL.

“PD-1” also includes any variant, derivative, or isoform of the PD-1gene or encoded protein. PD-1 is expressed on activated B cells, Tcells, and myeloid cells Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The initialmembers of the family, CD28 and ICOS, were discovered by functionaleffects on augmenting T cell proliferation following the addition ofmonoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansenet al. (1980) Immunogenics 10:247-260). Two ligands for PD-1 have beenidentified, PD-L1 and PD-L2, that have been shown to downregulate T cellactivation upon binding to PD-1 (Freeman et al. (2000) J Exp Med192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al.(2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologsthat bind to PD-1, but do not bind to other CD28 family members.

The complete human PD-L1 sequence can be found underUniProtKB/Swiss-Prot, identifier Q9NZQ7-1, shown as follows:

(SEQ ID NO: 3) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRINTTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTHLVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET.

PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat.Med. 8:787-9). The interaction between PD-1 and PD-L1 results in adecrease in tumor infiltrating lymphocytes, a decrease in T-cellreceptor mediated proliferation, and immune evasion by the cancerouscells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005)Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin.Cancer Res. 10:5094-100). Immune suppression can be reversed byinhibiting the local interaction of PD-1 with PD-L1, and the effect isadditive when the interaction of PD-1 with PD-L2 is blocked as well.

In the context of the present invention, “reduces the inhibitoryactivity of human PD-1”, “neutralizes PD-1” or “neutralizes theinhibitory activity of human PD-1” refers to a process in which PD-1 isinhibited in its signal transduction capacity resulting from theinteraction of PD-1 with one or more of its binding partners, such asPD-L1 or PD-L2. An agent that neutralizes the inhibitory activity ofPD-1 decreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of PD-1 with one or more ofits binding partners, such as PD-L1, PD-L2. Such an agent can therebyreduce the negative co-stimulatory signal mediated by or through cellsurface proteins expressed on T lymphocytes, so as to enhance T-celleffector functions such as proliferation, cytokine production and/orcytotoxicity.

Whenever “treatment of cancer” or the like is mentioned with referenceto an anti-NKG2A binding agent (e.g. antibody), are comprised: (a)method of treatment of cancer, said method comprising the step ofadministering (for at least one treatment) an NKG2A binding agent,(e.g., together or each separately in a pharmaceutically acceptablecarrier material) to an individual, a mammal, especially a human, inneed of such treatment, in a dose that allows for the treatment ofcancer, (a therapeutically effective amount), optionally in a dose(amount) as specified herein; (b) the use of an anti-NKG2A binding agentfor the treatment of cancer, or an anti-NKG2A binding agent, for use insaid treatment (especially in a human); (c) the use of an anti-NKG2Abinding agent for the manufacture of a pharmaceutical preparation forthe treatment of cancer, a method of using an anti-NKG2A binding agentfor the manufacture of a pharmaceutical preparation for the treatment ofcancer, comprising admixing an anti-NKG2A binding agent with apharmaceutically acceptable carrier, or a pharmaceutical preparationcomprising an effective dose of an anti-NKG2A binding agent that isappropriate for the treatment of cancer; or (d) any combination of a),b), and c), in accordance with the subject matter allowable forpatenting in a country where this application is filed.

The term “biopsy” as used herein is defined as removal of a tissue forthe purpose of examination, such as to establish diagnosis. Examples oftypes of biopsies include by application of suction, such as through aneedle attached to a syringe; by instrumental removal of a fragment oftissue; by removal with appropriate instruments through an endoscope; bysurgical excision, such as of the whole lesion; and the like.

The term “antibody,” as used herein, refers to polyclonal and monoclonalantibodies. Depending on the type of constant domain in the heavychains, antibodies are assigned to one of five major classes: IgA, IgD,IgE, IgG, and IgM. Several of these are further divided into subclassesor isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplaryimmunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids that is primarily responsible forantigen recognition. The terms variable light chain (V_(L)) and variableheavy chain (V_(H)) refer to these light and heavy chains respectively.The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are termed “alpha,” “delta,” “epsilon,”“gamma” and “mu,” respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG are the exemplary classes of antibodies employedherein because they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.Optionally the antibody is a monoclonal antibody. Particular examples ofantibodies are humanized, chimeric, human, or otherwise-human-suitableantibodies. “Antibodies” also includes any fragment or derivative of anyof the herein described antibodies.

The term “specifically binds to” means that an antibody can bindpreferably in a competitive binding assay to the binding partner, e.g.NKG2A, PD-1, PD-L1, as assessed using either recombinant forms of theproteins, epitopes therein, or native proteins present on the surface ofisolated target cells. Competitive binding assays and other methods fordetermining specific binding are well known in the art. For examplebinding can be detected via radiolabels, physical methods such as massspectrometry, or direct or indirect fluorescent labels detected using,e.g., cytofluorometric analysis (e.g. FACScan). Binding above the amountseen with a control, non-specific agent indicates that the agent bindsto the target. An agent that specifically binds NKG2A may bind NKG2Aalone or NKG2A as a dimer with CD94.

When an antibody is said to “compete with” a particular monoclonalantibody, it means that the antibody competes with the monoclonalantibody in a binding assay using either recombinant molecules (e.g.,NKG2A, PD-1, PD-L1) or surface expressed molecules (e.g., NKG2A, PD-1,PD-L1). For example, if a test antibody reduces the binding of anantibody having a heavy chain of any of SEQ ID NO: 4-8 and a light chainof SEQ ID NO: 9 to a NKG2A polypeptide or NKG2A-expressing cell in abinding assay, the antibody is said to “compete” respectively with suchantibody.

The term “affinity”, as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant Kd, defined as [Ab]×[Ag]/[Ab-Ag], where[Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant K_(a) isdefined by 1/Kd. Methods for determining the affinity of mAbs can befound in Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience, N.Y. (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein byreference. One standard method well known in the art for determining theaffinity of mAbs is the use of surface plasmon resonance (SPR) screening(such as by analysis with a BIAcore™ SPR analytical device).

Within the context herein a “determinant” designates a site ofinteraction or binding on a polypeptide.

The term “epitope” refers to an antigenic determinant, and is the areaor region on an antigen to which an antibody binds. A protein epitopemay comprise amino acid residues directly involved in the binding aswell as amino acid residues which are effectively blocked by thespecific antigen binding antibody or peptide, i.e., amino acid residueswithin the “footprint” of the antibody. It is the simplest form orsmallest structural area on a complex antigen molecule that can combinewith e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term ‘conformational’ istherefore often used interchangeably with ‘structural’.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials. The term “therapeutic agent” refers to anagent that has biological activity.

For the purposes herein, a “humanized” or “human” antibody refers to anantibody in which the constant and variable framework region of one ormore human immunoglobulins is fused with the binding region, e.g. theCDR, of an animal immunoglobulin. Such antibodies are designed tomaintain the binding specificity of the non-human antibody from whichthe binding regions are derived, but to avoid an immune reaction againstthe non-human antibody. Such antibodies can be obtained from transgenicmice or other animals that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge (see, e.g., Green etal. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856;Taylor et al. (1994) Int Immun 6:579, the entire teachings of which areherein incorporated by reference). A fully human antibody also can beconstructed by genetic or chromosomal transfection methods, as well asphage display technology, all of which are known in the art (see, e.g.,McCafferty et al. (1990) Nature 348:552-553). Human antibodies may alsobe generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos.5,567,610 and 5,229,275, which are incorporated in their entirety byreference).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The terms “Fc domain,” “Fc portion,” and “Fc region” refer to aC-terminal fragment of an antibody heavy chain, e.g., from about aminoacid (aa) 230 to about aa 450 of human γ (gamma) heavy chain or itscounterpart sequence in other types of antibody heavy chains (e.g., α,δ, ε and μ for human antibodies), or a naturally occurring allotypethereof. Unless otherwise specified, the commonly accepted Kabat aminoacid numbering for immunoglobulins is used throughout this disclosure(see Kabat et al. (1991) Sequences of Protein of Immunological Interest,5th ed., United States Public Health Service, National Institute ofHealth, Bethesda, MD).

The terms “isolated”, “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all.

Within the context herein, the term antibody that “binds” a polypeptideor epitope designates an antibody that binds said determinant withspecificity and/or affinity.

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Methods for determining identity are designed to give the largest matchbetween the sequences tested. Methods of determining identity aredescribed in publicly available computer programs. Computer programmethods for determining identity between two sequences include the GCGprogram package, including GAP (Devereux et al., Nucl. Acid. Res. 12,387 (1984); Genetics Computer Group, University of Wisconsin, Madison,Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215,403-410 (1990)). The BLASTX program is publicly available from theNational Center for Biotechnology Information (NCBI) and other sources(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschulet al., supra). The well-known Smith Waterman algorithm may also be usedto determine identity.

NKG2A-Neutralizing Therapeutic Agents

The anti-NKG2A agent binds an extra-cellular portion of human CD94/NKG2Areceptor and reduces the inhibitory activity of human CD94/NKG2Areceptor expressed on the surface of a CD94/NKG2A positive lymphocyte.In one embodiment the agent competes with HLA-E in binding toCD94/NKG2A, i.e. the agent blocks the interaction between CD94/NKG2A andits ligand HLA-E. In another embodiment the agent does not compete withHLA-E in binding to CD94/NKG2A; i.e. the agent is capable of bindingCD94/NKG2A simultaneously with HLA-E. The antibody may bind a combinedepitope on CD94 and NKG2A or and epitope on NKG2A alone.

In one aspect the anti-NKG2A agent is an antibody selected from a fullyhuman antibody, a humanized antibody, and a chimeric antibody. In oneaspect, the agent comprises a constant domain derived from a human IgG1,IgG2, IgG3 or IgG4 antibody. In one aspect, the agent is a fragment ofan antibody selected from IgA, an IgD, an IgG, an IgE and an IgMantibody. In one aspect, the agent is an antibody fragment selected froma Fab fragment, a Fab′ fragment, a Fab′-SH fragment, a F(ab)2 fragment,a F(ab′)2 fragment, an Fv fragment, a Heavy chain Ig (a llama or camelIg), a VHH fragment, a single domain FV, and a single-chain antibodyfragment. In one aspect, the agent is a synthetic or semisyntheticantibody-derived molecule selected from a scFV, a dsFV, a minibody, adiabody, a triabody, a kappa body, an IgNAR, and a multispecificantibody.

Optionally, the anti-NKG2A antibodies do not demonstrate substantialspecific binding to human Fcγ receptors, e.g. CD16. Optionally, theanti-NKG2A antibodies lack substantial specific binding or have low ordecreased specific binding to one or more, or all of, human CD16, CD32A,CD32B or CD64. Exemplary antibodies may comprise constant regions ofvarious heavy chains that are known not to bind or to have low bindingto Fcγ receptors. One such example is a human IgG4 constant region. Inone embodiment, the IgG4 antibody comprises a modification to preventthe formation of half antibodies (fab arm exchange) in vivo, e.g., theantibody comprises an IgG4 heavy chain comprising a serine to prolinemutation in residue 241, corresponding to position 228 according to theEU-index (Kabat et al., “Sequences of proteins of immunologicalinterest”, 5^(th) ed., NIH, Bethesda, M L, 1991). Such modified IgG4antibodies will remain intact in vivo and maintain a bivalent (highaffinity) binding to NKG2A, as opposed to native IgG4 that will undergofab arm exchange in vivo such that they bind to NKG2A in monovalentmanner which can alter binding affinity. Alternatively, antibodyfragments that do not comprise constant regions, such as Fab or F(ab′)2fragments, can be used to avoid Fc receptor binding. Fc receptor bindingcan be assessed according to methods known in the art, including forexample testing binding of an antibody to Fc receptor protein in aBIACORE assay. Also, any human antibody type (e.g. IgG1, IgG2, IgG3 orIgG4) can be used in which the Fc portion is modified to minimize oreliminate binding to Fc receptors (see, e.g., WO03101485, the disclosureof which is herein incorporated by reference). Assays such as, e.g.,cell based assays, to assess Fc receptor binding are well known in theart, and are described in, e.g., WO03101485.

The present invention thus concerns antibodies or other agents bindingto NKG2A. In one aspect, the antibody binds to NKG2A with a KD at least100-fold lower than to human NKG2C and/or NKG2E.

In one aspect of the invention, the agent reduces CD94/NKG2A-mediatedinhibition of a CD94/NKG2A-expressing lymphocyte by interfering withCD94/NKG2A signalling by, e.g., interfering with the binding of HLA-E byNKG2A, preventing or inducing conformational changes in the CD94/NKG2Areceptor, and/or affecting dimerization and/or clustering of theCD94/NKG2A receptor.

In one aspect of the invention, the agent binds to an extracellularportion of NKG2A with a KD at least 100 fold lower than to NKG2C. In afurther preferred aspect, the agent binds to an extracellular portion ofNKG2A with a KD at least 150, 200, 300, 400, or 10,000 fold lower thanto NKG2C. In another aspect of the invention, the agent binds to anextracellular portion of NKG2A with a KD at least 100 fold lower than toNKG2C, NKG2E and/or NKG2H molecules. In a further preferred aspect, theagent binds to an extracellular portion of NKG2A with a KD at least 150,200, 300, 400, or 10,000 fold lower than to NKG2C, NKG2C and/or NKG2Hmolecules. This can be measured, for instance, in BiaCore experiments,in which the capacity of agents to bind the extracellular portion ofimmobilized CD94/NKG2A (e.g. purified from CD94/NKG2 expressing cells,or produced in a bio-system) is measured and compared to the binding ofagents to similarly produced CD94/NKG2C and/or other CD94/NKG2 variantsin the same assay. Alternatively, the binding of agents to cells thateither naturally express, or over-express (e.g. after transient orstable transfection), CD94/NKG2A can be measured and compared to bindingof cells expressing CD94/NKG2C and/or other CD94/NKG2 variants.Anti-NKG2A antibodies may optionally bind NKG2B, which is an NKG2Asplice variant forming an inhibitory receptor together with CD94. In oneembodiment, affinity can be measured using the methods disclosed in U.S.Pat. No. 8,206,709, for example by assessing binding to covalentlyimmobilized NKG2A-CD94-Fc fusion protein by Biacore as shown in Example8 of U.S. Pat. No. 8,206,709, the disclosure of which is incorporateherein by reference.

The anti-NKG2A antibody can be a humanized antibody, for examplecomprising a VH human acceptor framework from a human acceptor sequenceselected from, e.g., VH1_18, VH5_a, VH5_51, VH1_f, and VH1_46, and a JH6J-segment, or other human germline VH framework sequences known in theart. The VL region human acceptor sequence may be, e.g., VKI_02/JK4.

In one embodiment, the antibody is a humanized antibody based onantibody Z270. Different humanized Z270VH chains are shown in SEQ IDNOS: 4-8 (variable region domain amino acid underlined). HumZ270VH6 (SEQID NO: 4) is based on VH5_51; HumZ270VH1 (SEQ ID NO: 5) is based onVH1_18; humZ270VH5 (SEQ ID NO: 6) is based on VH5_a; humZ270VH7 (SEQ IDNO: 7) is based on VH1_f; and humZ270VH8 (SEQ ID NO: 8) is based onVH1_46; all with a JH6 J-segment. Each of these antibodies retains highaffinity binding to NKG2A, with low likelihood of a host immune responseagainst the antibody as the 6 C-terminal amino acid residues of theKabat CDR-H2 of each of the humanized constructs are identical to thehuman acceptor framework. Using the alignment program VectorNTl, thefollowing sequence identities between humZ270VH1 and humZ270VH5, -6, -7,and -8 were obtained: 78.2% (VH1 vs. VH5), 79.0% (VH1 vs. VH6), 88.7%(VH1 vs. VH7), and 96.0% (VH1 vs. VH8).

In one aspect, the agent comprises (i) a heavy chain variable region ofany of SEQ ID NOS: 4-8, or an amino acid sequence at least 50%, 60%,70%, 80%, 90%, 95%, 98% or 99% identical thereto, and (ii) a light chainvariable region of SEQ ID NO: 9, or an amino acid sequence at least 50%,60%, 70%, 80%, 90%, 95%, 98% or 99% identical thereto. In one aspect,the agent comprises (i) a heavy chain comprising the amino acid sequenceof any of SEQ ID NOS: 4-8, or an amino acid sequence at least 50%, 60%,70%, 80%, 90%, 95%, 98% or 99% identical thereto, and (ii) a light chaincomprising the amino acid sequence of SEQ ID NO: 9, or an amino acidsequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identicalthereto. The antibody having the heavy chain of any of SEQ ID NOS: 4-8and a light chain of SEQ ID NO: 9 neutralizes the inhibitory activity ofNKG2A, but does not substantially bind the activating receptors NKG2C,NKGE or NKG2H. This antibody furthermore competes with HLA-E for bindingto NKG2A on the surface of a cell. In one aspect, the agent comprisesHCDR1, HCDR2 and/or HCDR3 sequences derived from the heavy chain havingthe amino acid sequence of any of SEQ ID NO: 4-8. In one aspect of theinvention, the agent comprises LCDR1, LCDR2 and/or LCDR3 sequencesderived from the light chain having the amino acid sequence of SEQ IDNO: 9.

Heavy Chains VH6: (SEQ ID NO: 4)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK VH1: (SEQ ID NO: 5)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK VH5: (SEQ ID NO: 6)EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWMNWVRQMPGKGLEWMGRIDPYDSETHYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK VH7: (SEQ ID NO: 7)EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMNWVQQAPGKGLEWMGRIDPYDSETHYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK VH8: (SEQ ID NO: 8)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Light chain (SEQ ID NO: 9)DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

In one aspect, the anti-NKG2A antibody is an antibody comprising aCDR-H1 corresponding to residues 31-35 of SEQ ID NOS: 4-8, a CDR-H2corresponding to residues 50-60 (optionally 50-66 when including aminoacids of human origin) of SEQ ID NOS: 4-8, and a CDR-H3 corresponding toresidues 99-114 (95-102 according to Kabat) of SEQ ID NOS: 4-8. In oneembodiment, the CDR-H2 corresponding to residues 50-66 of SEQ ID NOS:4-8. Optionally, a CDR may comprise one, two, three, four, or more aminoacid substitutions.

In one aspect, the anti-NKG2A antibody is an antibody comprising aCDR-L1 corresponding to residues 24-34 of SEQ ID NO: 9, a CDR-L2corresponding to residues 50-56 of SEQ ID NO: 9, and an CDR-L3corresponding to residues 89-97 of SEQ ID NO: 9. Optionally, a CDR maycomprise one, two, three, four, or more amino acid substitutions.

In one aspect, the anti-NKG2A antibody is an antibody comprising aCDR-H1 corresponding to residues 31-35 of SEQ ID NOS: 4-8, a CDR-H2corresponding to residues 50-60 (optionally 50-66) of SEQ ID NOS: 4-8,and a CDR-H3 corresponding to residues 99-114 (95-102 according toKabat) of SEQ ID NOS: 4-8, a CDR-L1 corresponding to residues 24-34 ofSEQ ID NO: 9, a CDR-L2 corresponding to residues 50-56 of SEQ ID NO: 9,and an CDR-L3 corresponding to residues 89-97 of SEQ ID NO: 9.

In one aspect, the agent comprises HCDR1, HCDR2 and/or HCDR3 sequencesderived from the VH having the amino acid sequence of SEQ ID NO: 10. Inone aspect of the invention, the agent comprises LCDR1, LCDR2 and/orLCDR3 sequences derived from the VL having the amino acid sequence ofSEQ ID NO: 11. In one aspect, the agent comprises HCDR1, HCDR2 and/orHCDR3 sequences derived from the VH having the amino acid sequence ofSEQ ID NO: 10, and LCDR1, LCDR2 and/or LCDR3 sequences derived from theVL having the amino acid sequence of SEQ ID NO: 11. The antibody havingthe heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 11neutralizes the inhibitory activity of NKG2A, and also binds theactivating receptors NKG2C, NKG2E or NKG2H. The antibody does notcompetes with HLA-E for binding to NKG2A on the surface of a cell (i.e.it is a non-competitive antagonist of NKG2A).

(SEQ ID NO: 10) EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEISSLRSEDTAMYYCTRHGDY PRFFDVWGAGTTVTVSS(SEQ ID NO: 11) QIVLTQSPALMSASPGEKVTMTCSASSSVSYIYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSGNPYTFGGGTK LEIKR

In one aspect, the agent comprises amino acid residues 31-35, 50-60, 62,64, 66, and 99-108 of the variable-heavy (VH) domain (SEQ ID NO: 10) andamino acid residues 24-33, 49-55, and 88-96 of the variable-light (VL)domain (SEQ ID NO: 11), optionally with one, two, three, four, or moreamino acid substitutions.

In one aspect, the agent is a fully human antibody which has been raisedagainst the CD94/NKG2A epitope to which any of the aforementionedantibodies bind.

It will be appreciated that, while the aforementioned antibodies can beused, other antibodies can recognize and be raised against any part ofthe NKG2A polypeptide so long as the antibody causes the neutralizationof the inhibitory activity of NKG2A. For example, any fragment of NKG2A,preferably but not exclusively human NKG2A, or any combination of NKG2Afragments, can be used as immunogens to raise antibodies, and theantibodies can recognize epitopes at any location within the NKG2Apolypeptide, so long as they can do so on NKG2A expressing NK cells asdescribed herein. Optionally, the epitope is the epitope specificallyrecognized by antibody having the heavy chain of SEQ ID NOS: 4-8 and thelight chain of SEQ ID NO: 9.

In one aspect, the agent competes with humZ270 antibody disclosed inU.S. Pat. No. 8,206,709 (the disclosure of which is incorporated hereinby reference) in binding to the extra-cellular portion of humanCD94/NKG2A receptor. Competitive binding can be measured, for instance,in BiaCore experiments, in which the capacity of agents is measured, forbinding the extracellular portion of immobilized CD94/NKG2A receptor(e.g. purified from CD94/NKG2 expressing cells, or produced in abio-system) saturated with humZ270. Alternatively, the binding of agentsto cells is measured that either naturally express, or over-express(e.g. after transient or stable transfection), CD94/NKG2A receptor, andwhich have been pre-incubated with saturating doses of Z270. In oneembodiment, competitive binding can be measured using the methodsdisclosed in U.S. Pat. No. 8,206,709, for example by assessing bindingto Ba/F3-CD94-NKG2A cells by flow cytometry as shown in Example 15 ofU.S. Pat. No. 8,206,709, the disclosure of which is incorporate hereinby reference.

PD-1 Neutralizing Therapeutic Agents

There are currently at least six agents blocking the PD-1/PD-L1 pathwaythat are marketed or in clinical evaluation. One agent is BMS-936558(Nivolumab/ONO-4538, Bristol-Myers Squibb; formerly MDX-1106).Nivolumab, (Trade name Opdivo®) is an FDA-approved fully human IgG4anti-PD-L1 mAb that inhibits the binding of the PD-L1 ligand to bothPD-1 and CD80 and is described as antibody 5C4 in WO 2006/121168, thedisclosure of which is incorporated herein by reference. For melanomapatients, the most significant OR was observed at a dose of 3 mg/kg,while for other cancer types it was at 10 mg/kg. Nivolumab is generallydosed at 10 mg/kg every 3 weeks until cancer progression.

MK-3475 (human IgG4 anti-PD1 mAb from Merck), also referred to aslambrolizumab or pembrolizumab (Trade name Keytruda®) has been approvedby the FDA for the treatment of melanoma and is being tested in othercancers. Pembrolizumab was tested at 2 mg/kg or 10 mg/kg every 2 or 3weeks until disease progression. DNA constructs encoding the variableregions of the heavy and light chains of the humanized antibodiesh409All have been deposited with the American Type Culture CollectionPatent Depository (10801 University Blvd., Manassas, VA). The plasmidcontaining the DNA encoding the heavy chain of h409A-I 1 was depositedon Jun. 9, 2008 and identified as 081469_SPD-H and the plasmidcontaining the DNA encoding the light chain of h409Al 1 was deposited onJun. 9, 2008 and identified as 0801470_SPD-L-I 1. MK-3475, also known asMerck 3745 or SCH-900475, is also described in WO2009/114335.

MPDL3280A/RG7446 (anti-PD-L1 from Roche/Genentech) is a human anti-PD-L1mAb that contains an engineered Fc domain designed to optimize efficacyand safety by minimizing FcγR binding and consequentialantibody-dependent cellular cytotoxicity (ADCC). Doses of ≤10, 15, and25 mg/kg MPDL3280A were administered every 3 weeks for up to 1 year. Inphase 3 trial, MPDL3280A is administered at 1200 mg by intravenousinfusion every three weeks in NSCLC.

AMP-224 (Amplimmune and GSK) is an immunoadhesin comprising a PD-L2extracellular domain fused to an Fc domain. Other examples of agentsthat neutralize PD-1 may include an antibody that binds PD-L2 (ananti-PD-L2 antibody) and blocks the interaction between PD-1 and PD-L2.

Pidlizumab (CT-011; CureTech) (humanized IgG1 anti-PD1 mAb fromCureTech/Teva), Pidlizumab (CT-011; CureTech) (see e.g., WO2009/101611)Thirty patients with rituximab-sensitive relapsed FL were treated with 3mg/kg intravenous CT-011 every 4 weeks for 4 infusions in combinationwith rituximab dosed at 375 mg/m2 weekly for 4 weeks, starting 2 weeksafter the first infusion of CT-011.

Further known PD-1 antibodies and other PD-1 inhibitors include AMP-224(a B7-DC/IgG1 fusion protein licensed to GSK), AMP-514 described in WO2012/145493, antibody MEDI-4736 (an anti-PD-L1 developed byAstraZeneca/Medimmune) described in WO2011/066389 and US2013/034559,antibody YW243.55.S70 (an anti-PD-L1) described in WO2010/077634,MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody developedby Bristol-Myers Squibb described in WO2007/005874, and antibodies andinhibitors described in WO2006/121168, WO2009/014708, WO2009/114335 andWO2013/019906, the disclosures of which are hereby incorporated byreference. Further examples of anti-PD1 antibodies are disclosed inWO2015/085847 (Shanghai Hengrui Pharmaceutical Co. Ltd.), for exampleantibodies having light chain variable domain CDR1, 2 and 3 of SEQ IDNO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8, respectively, and antibodyheavy chain variable domain CDR1, 2 and 3 of SEQ ID NO: 3, SEQ ID NO: 4or SEQ ID NO: 5, respectively, wherein the SEQ ID NO references are thenumbering according to WO2015/085847, the disclosure of which isincorporated herein by reference. Antibodies that compete with any ofthese antibodies for binding to PD-1 or PD-L1 also can be used.

An exemplary anti-PD-1 antibody is pembrolizumab (see, e.g., WO2009/114335 the disclosure of which is incorporated herein byreference.). The anti-PD-1 antibody may be the antibody h409Al 1 in WO2008/156712, comprising heavy chain variable regions encoded by the DNAdeposited at the ATCC as 081469_SPD-H and light chain variable regionsencoded by the DNA deposited at the ATCC as 0801470_SPD-L-I 1. In otherembodiments, the antibody comprises the heavy and light chain CDRs orvariable regions of pembrolizumab. Accordingly, in one embodiment, theantibody comprises the CDR1, CDR2, and CDR3 domains of the VH ofpembrolizumab encoded by the DNA deposited at the ATCC as 081469_SPD-H,and the CDR1, CDR2 and CDR3 domains of the VL of pembrolizumab encodedby the DNA deposited at the ATCC as 0801470_SPD-L-I 1.

In some embodiments, the PD-1 neutralizing agent is an anti-PD-L1 mAbthat inhibits the binding of PD-L1 to PD-1. In some embodiments, thePD-1 neutralizing agent is an anti-PD1 mAb that inhibits the binding ofPD-1 to PD-L1. In some embodiments, the PD-1 neutralizing agent is animmunoadhesin (e.g., an immunoadhesin comprising an extracellular orPD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g.,an Fc region of an immunoglobulin sequence).

Another exemplary anti-PD-1 antibody is nivolumab comprising heavy andlight chains having the respective sequences shown in SEQ ID NOs: 12 and13 or a respective amino acid sequence at least 50%, 60%, 70%, 80%, 90%,95%, 98% or 99% identical thereto, or antigen binding fragments andvariants thereof. In other embodiments, the antibody comprises the heavyand light chain CDRs or variable regions of nivolumab. Accordingly, inone embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domainsof the heavy chain of nivolumab having the sequence set forth in SEQ IDNO: 12, and the CDR1, CDR2 and CDR3 domains of the light chain ofnivolumab having the sequences set forth in SEQ ID NO: 13.

(SEQ ID NO: 12) QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVrWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT TYTCNVDHKPSNTKVD RVES YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYYDGVEVHNA TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEKNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. (SEQ ID NO: 13)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEI RTVAAPSVFI FPPSDEQL SGTASVVCLLNNFYPREA VQWKVDNALQSGNSQESVTEQDS DSTYSL SSTLLSKADYEKHKVYACEVTHQGLSS PVTSFNRGEC.

An exemplary anti-PD-L1 antibody comprises heavy and light chainvariable regions having the respective sequences shown in SEQ ID NOs: 14and 15, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%,98% or 99% identical thereto respectively, or antigen binding fragmentsand variants thereof. In other embodiments, the antibody comprises theheavy and light chain CDRs or variable regions of MPDL3280A.Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2,and CDR3 domains of the heavy chain having the sequence set forth in SEQID NO: 14, and the CDR1, CDR2 and CDR3 domains of the light chain havingthe sequences set forth in SEQ ID NO: 15.

(SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQ NSLRAEDTAVYYCARRHWP GGFDYWG QGTLVTVSS(SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQ PGKAPKLLIY SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQG TKVEIKR

The anti-PD-1 or anti-PD-L1 antibody can be selected from a fully humanantibody, a humanized antibody, and a chimeric antibody. In one aspectof the invention, the agent comprises a constant domain derived from ahuman IgG1, IgG2, IgG3 or IgG4 antibody. In one aspect of the invention,the agent is a fragment of an antibody selected from IgA, an IgD, anIgG, an IgE and an IgM antibody. In one aspect of the invention, theagent is an antibody fragment selected from a Fab fragment, a Fab′fragment, a Fab′-SH fragment, a F(ab)2 fragment, a F(ab′)2 fragment, anFv fragment, a Heavy chain Ig (a llama or camel Ig), a VHH fragment, asingle domain FV, and a single-chain antibody fragment. In one aspect ofthe invention, the agent is a synthetic or semisyntheticantibody-derived molecule selected from a scFV, a dsFV, a minibody, adiabody, a triabody, a kappa body, an IgNAR; and a multispecificantibody.

The anti-PD-1 or anti-PD-L1 antibody can lack substantial specificbinding to Fey receptors, e.g. CD16. Such antibodies may compriseconstant regions of various heavy chains that are known not to bind Fcreceptors. One such example is an IgG4 constant region. IgG4Alternatively, antibody fragments that do not comprise constant regions,such as Fab or F(ab′)2 fragments, can be used to avoid Fc receptorbinding. Fc receptor binding can be assessed according to methods knownin the art, including for example testing binding of an antibody to Fcreceptor protein in a BIACORE assay. Also, any human antibody type (e.g.IgG1, IgG2, IgG3 or IgG4) can be used in which the Fc portion ismodified to minimize or eliminate binding to Fcγ receptors. Theanti-PD-1 or anti-PDL1 antibody, the antibody will therefore typicallyhave reduced or minimal effector function. In one aspect, the minimaleffector function results from production in prokaryotic cells. In oneaspect the minimal effector function results from an “effector-less Fcmutation” or aglycosylation. In still a further embodiment, theeffector-less Fc mutation is an N297A or D265A/N297A substitution in theconstant region.

An anti-NKG2A or anti-PD-1 or anti-PD-L1 agent such as an antibody canbe incorporated in a pharmaceutical formulation in a concentration from1 mg/ml to 500 mg/ml, wherein said formulation has a pH from 2.0 to10.0. The formulation may further comprise a buffer system,preservative(s), tonicity agent(s), chelating agent(s), stabilizers andsurfactants. In one embodiment, the pharmaceutical formulation is anaqueous formulation, i.e., formulation comprising water. Suchformulation is typically a solution or a suspension. In a furtherembodiment, the pharmaceutical formulation is an aqueous solution. Theterm “aqueous formulation” is defined as a formulation comprising atleast 50% w/w water. Likewise, the term “aqueous solution” is defined asa solution comprising at least 50% w/w water, and the term “aqueoussuspension” is defined as a suspension comprising at least 50% w/wwater.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment, the pharmaceutical formulation is a driedformulation (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of such an antibody, and a buffer, wherein the antibody ispresent in a concentration from 1 mg/ml or above, and wherein saidformulation has a pH from about 2.0 to about 10.0.

In a another embodiment, the pH of the formulation is in the rangeselected from the list consisting of from about 2.0 to about 10.0, about3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, andabout 5.5 to about 7.5.

In a further embodiment, the buffer is selected from the groupconsisting of sodium acetate, sodium carbonate, citrate, glycylglycine,histidine, glycine, lysine, arginine, sodium dihydrogen phosphate,disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment, the formulation further comprises apharmaceutically acceptable preservative. In a further embodiment, theformulation further comprises an isotonic agent. In a furtherembodiment, the formulation also comprises a chelating agent. In afurther embodiment of the invention the formulation further comprises astabilizer. In a further embodiment, the formulation further comprises asurfactant. For convenience reference is made to Remington: The Scienceand Practice of Pharmacy, 19th edition, 1995.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Administration of pharmaceutical compositions according to the inventionmay be through several routes of administration, for example,intravenous. Suitable antibody formulations can also be determined byexamining experiences with other already developed therapeuticmonoclonal antibodies. Several monoclonal antibodies have been shown tobe efficient in clinical situations, such as Rituxan (Rituximab),Herceptin (Trastuzumab) Xolair (Omalizumab), Bexxar (Tositumomab),Campath (Alemtuzumab), Zevalin, Oncolym and similar formulations may beused with the antibodies of this invention. For example, a monoclonalantibody can be supplied at a concentration of 10 mg/mL in either 100 mg(10 mL) or 500 mg (50 mL) single-use vials, formulated for IVadministration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citratedihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection.The pH is adjusted to 6.5. In another embodiment, the antibody issupplied in a formulation comprising about 20 mM Na-Citrate, about 150mM NaCl, at pH of about 6.0.

Also provided are kits which include a pharmaceutical compositioncontaining an anti-NKG2A antibody, and optionally further an anti-PD-1or anti-PD-L1 antibody, and a pharmaceutically-acceptable carrier, in atherapeutically effective amount adapted for use in the precedingmethods. The kits optionally also can include instructions, e.g.,comprising administration schedules, to allow a practitioner (e.g., aphysician, nurse, or patient) to administer the composition containedtherein to administer the composition to a patient having cancer (e.g.,a solid tumor). The kit also can include a syringe.

Optionally, the kits include multiple packages of the single-dosepharmaceutical compositions each containing an effective amount of theanti-NKG2A, and optionally further an anti-PD-1 or PD-L1 antibody, for asingle administration in accordance with the methods provided above.Instruments or devices necessary for administering the pharmaceuticalcomposition(s) also may be included in the kits. For instance, a kit mayprovide one or more pre-filled syringes containing an amount of theanti-NKG2A, anti-PD-1 or anti-PD-L1 antibody.

In one embodiment, the present invention provides a kit for treating ananti-PD-1/PD-L1 antibody resistant cancer in a human patient, the kitcomprising:

-   -   (a) a dose of an anti-NKG2A antibody comprising the CDR1, CDR2        and CDR3 domains of a heavy chain having the sequence set forth        in any of SEQ ID NOS: 4-8, and the CDR1, CDR2 and CDR3 domains        of a light chain having the sequence set forth in SEQ ID NO: 9;    -   (b) optionally, a dose of an anti-PD-1 antibody or an anti-PD-L1        antibody; and    -   (c) optionally, instructions for using the anti-NKG2A antibody        (and optionally anti-PD-1 or PD-L1 antibody) in any of the        methods described herein.

Diagnostics, Prognostics, and Treatment of Malignancies

Described are methods useful in the diagnosis, prognosis, monitoring,treatment and prevention of a cancer in an individual with an agent thatneutralizes the activity of NKG2A, optionally further in combinationwith an agent that neutralizes the activity of PD-1, e.g. an anti-PD-1antibody or an anti-PD-L1 antibody. An individual may optionally be apoor responder to treatment with an agent that neutralizes theinhibitory activity of PD-1, for example, an individual who experiencesor is predicted to have a high likelihood to experience (e.g. based onone or more prognostic factors) an incomplete response, lack oftherapeutic response, detectable or residual cancer and/or progressivedisease upon (during or following) treatment with an agent thatneutralizes the inhibitory activity of PD-1. In one embodiment, theindividual's cancer has not experienced a complete response or ispredicted to have a likelihood (e.g. a high likelihood) not toexperience a complete response upon (during or following) treatment withan agent that neutralizes the inhibitory activity of PD-1. In oneembodiment, the individual's cancer has progressed (e.g. progressivedisease) upon (during or following) treatment with an agent thatneutralizes the inhibitory activity of PD-1. In one embodiment, theindividual's cancer has partially responded or stabilized (partialresponse or stable disease) but is predicted to have a likelihood toprogress upon (during or following) treatment with an agent thatneutralizes the inhibitory activity of PD-1.

In one example, the individual has a cancer known to be poorlyresponsive to treatment with an agent that neutralizes the inhibitoryactivity of PD-1 (e.g. in monotherapy). In one example, the individualhas a cancer known to be characterized by tumor infiltratingNKG2A-expressing CD8+ or NK cells upon treatment with an agent thatneutralizes the inhibitory activity of PD-1 (e.g. in monotherapy). Inone example, the individual has a cancer known to be characterized byexpression (or increased expression) of HLA-E on cancer cells upontreatment with an agent that neutralizes the inhibitory activity of PD-1(e.g. in monotherapy). Optionally the cancer is a head and neck squamouscell carcinoma, a non-small cell lung cancer (NSCLC), kidney cancer,gastrointestinal cancer, pancreatic or esophagus adenocarcinoma, breastcancer, renal cell carcinoma (RCC), melanoma, colorectal cancer orovarian cancer. Such an individual can advantageously be treated with anagent that neutralizes the inhibitory activity of NKG2A in combinationwith an agent that neutralizes the inhibitory activity of PD-1.

For example, the individual may have a cancer that is poorly responsive(or resistant or non-responsive), for example a cancer that has relapsedor progressed despite (e.g. during or following) treatment with an agentthat neutralizes the inhibitory activity of PD-1. In one embodiment, theindividual treated with an anti-NKG2A agent has experienced anincomplete response (has not experienced a complete response (CR)) upon(during or following) treatment with an agent that neutralizes theinhibitory activity of PD-1 (e.g. as monotherapy or as combinationtherapy with an agent other than an agent that neutralizes NKG2A), orhas experienced at least a partial response (PR) upon (during orfollowing) treatment with an agent that neutralizes the inhibitoryactivity of PD-1, but whose cancer has relapsed or progressed. In anyembodiment herein, treatment response can be defined and/or assessedaccording to well-known criteria, e.g. Response Evaluation Criteria InSolid Tumors (RECIST), such as version 1.1, see Eisenhauer et al. (2009)Eur. J. Cancer 45:228-247, or Immune-Related Response Criteria (irRC),see Wolchock et al. (2009) Clinical Cancer Research 15:7412-7420.

In one embodiment, an individual who is a poor responder (or who has acancer that is poorly responsive) is an individual having a poor diseaseprognosis for treatment with an agent that neutralizes the inhibitoryactivity of PD-1. An individual having a poor disease prognosis can, forexample, be determined to have a high or higher risk of cancerprogression (e.g. compared to individuals having a good diseaseprognostic), based on one or more predictive factors. In one embodiment,a predictive factor(s) comprises presence of elevated numbers ofNKG2A-expressing NK and/or CD8 T cells and/or elevated levels of NKG2Aon NK and/or CD8 T cells can indicate an individual has a poor prognosisfor response to treatment with an antibody that neutralizes PD-1. In oneembodiment, a predictive factor(s) comprises presence or absence of amutation in one or more genes. In one embodiment, the mutation defines aneo-epitope recognized by a T cell. In one embodiment, the predictivefactor(s) comprises level(s) of expression of one or more genes orproteins in tumor cells, e.g. PD-L1, decreased or elevated levels ofPD-L1 on tumor cells. In one embodiment, the predictive factor(s)comprises level(s) of expression of one or more genes or proteins in NKand/or CD8 T cells in circulation or in the tumor environment, e.g.,PD-1. In one embodiment, the predictive factor(s) comprises mutationalload in cancer cells, e.g. number of non-synonymous mutations per exome.

The treatment regimens and methods described herein may be used with orwithout a prior step of detecting the expression PD-L1 on cells in abiological sample obtained from an individual (e.g. a biological samplecomprising cancer cells, cancer tissue or cancer-adjacent tissue). Inanother embodiment, the disclosure provides a method for the treatmentor prevention of a cancer in an individual in need thereof, the methodcomprising:

-   -   a) detecting cells (e.g. tumor cells, tumor infiltrating immune        cells, tumor infiltrating macrophages) in a sample from the        individual that express PD-L1, and    -   b) upon a determination that cells which express PD-L1 are        comprised in the sample, optionally at a reference level that        corresponds to an individual poorly responsive and/or not        deriving substantial benefit from an agent that neutralizes the        inhibitory activity of PD-1, administering to the individual an        agent that neutralizes the inhibitory activity of NKG2A,        optionally in combination with an agent that neutralizes the        inhibitory activity of PD-1. The PD-L1 reference level can be        characterized by any suitable conventionally used reference        level. For example, if 1% or less, optionally 5% or less,        optionally 10% or less, optionally 50% or less of tumor cells or        cells from a tumor tissue sample express PD-L1 (e.g. using an        immunohistochemistry-based assay), the sample can be determine        to correspond to an individual poorly responsive and/or not        deriving substantial benefit from an agent that neutralizes the        inhibitory activity of PD-1. Example of such assays include the        PD-L1 IHC 22C3 assay from pharmDx from Dako Denmark A/S. In this        assay, PD-L1 expression level is measured using the tumor        proportion score (TPS), the percentage of tumor cells staining        for PD-L1 (0% to 100%). Optionally, a reference level is a level        for non-high PD-L1 expression, optionally wherein less 50% tumor        cells express PD-L1 (e.g., the patients have a TPS of less than        50%).

The treatment regimens and methods described herein can be useful forthe treatment of solid tumors and hematological cancers. The methods andcompositions of the present invention are utilized for example thetreatment of a variety of cancers and other proliferative diseasesincluding, but not limited to: squamous cell cancers, carcinomas,including that of the bladder, breast, colon, kidney, liver, lung,ovary, head and neck, prostate, pancreas, stomach, cervix, thyroid andskin; hematopoietic tumors of lymphoid lineage, including leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkettslymphoma, and multiple myeloma; hematopoietic tumors of myeloid lineage,including acute and chronic myelogenous leukemias, promyelocyticleukemia, and myelodysplastic syndrome; tumors of mesenchymal origin,including fibrosarcoma and rhabdomyosarcoma; other tumors, includingmelanoma, seminoma, terato-carcinoma, neuroblastoma and glioma; tumorsof the central and peripheral nervous system, including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoacanthoma,seminoma, and thyroid follicular cancer.

In one embodiment, the cancer is a head and neck squamous cell carcinoma(HNSCC). In one embodiment the HNSCC is an oropharangeal tumor, a larynxtumor, a tumor of the oral cavity, or a tumor of the hypopharynx. In oneembodiment, the HNSCC is an oral cavity SCC (OCSCC). OCSCC comprisessquamous cell carcinoma of the lip, of the anterior ⅔ of the tongue,floor of the mouth, buccal mucosa, gingiva, hard palate and retromolartrigone. In one embodiment the HNSCC is a metastatic cancer.

When treating an individual having a solid tumor, a compound (e.g.antibody) that neutralizes the inhibitory activity of a human NKG2Apolypeptide can advantageously be administered according to a treatmentregimen described herein, to an individual having a cancer who has notreceived surgery to remove cancer cells, or who has not in the currentperiod received such surgery. However it will be appreciated that thecompound can also be administered to a patient who has received, or whois undergoing, surgery to remove cancer cells. Where the anti-NKG2Acompound is administered to an individual who has not received surgicalintervention to remove cancer cells (e.g. to remove HNSCC cells), theNKG2A-binding compound can for example be administered approximately 1to 8 weeks prior to surgery. In one embodiment, at least one (e.g. one,two, three or more) complete ad-ministration cycle(s) of treatment withanti-NKG2A compound is administered prior to surgery. In one embodiment,the administration cycle is between 2 weeks and 8 week.

Therapies for the treatment of a PD-1/PD-L1 poorly responsive cancerprovided herein involve administration of a neutralizing anti-NKG2Aantibody, optionally in the absence or optionally in combination with aPD-1-neutralizing agent, e.g. a neutralizing anti-PD-1 or anti-PD-L1antibody, to treat subjects afflicted with cancer (e.g., advancedrefractory or progressing solid or hematological tumors). In oneembodiment, the invention provides an anti-NKG2A antibody, andoptionally further an anti-PD-1 antibody in combination, to treatsubjects having a solid tumor (e.g., a solid tumor, an advancedrefractory solid tumor) or subjects having a hematological tumor. In aparticular embodiment, the anti-NKG2A antibody comprises a heavy chainof any of SEQ ID NOS: 4-8 and a light chain of SEQ ID NO: 9. In oneembodiment, the antibody that neutralizes the inhibitory activity ofPD-1 is selected from the group consisting of pembrolizumab, nivolumab,AMP-514, MEDI-4736, CT-011 and MPDL3280A.

As used herein, adjunctive or combined administration(co-administration) includes simultaneous administration of thecompounds in the same or different dosage form, or separateadministration of the compounds (e.g., sequential administration). Thus,the anti-NKG2A and anti-PD-1 or anti-PD-L1 antibodies can besimultaneously administered in a single formulation. Alternatively, theanti-NKG2A and anti-PD-1 or anti-PD-L1 antibodies can be formulated forseparate administration and are administered concurrently orsequentially.

In one embodiment, the cancer treated with the methods disclosed hereinis a cancer characterized by infiltration of NK cells and/or CD8 T cellsexpressing at their surface NKG2A. In one embodiment, the cancer treatedwith the methods disclosed herein is a cancer characterized byinfiltration of NK cells, wherein at least 20%, 30%, 40% or 50% of theNK cells express at their surface NKG2A.

In one embodiment, the cancer treated with the methods disclosed hereinis a cancer characterized by high levels of HLA-E. In one embodiment,the cancer is selected from the group consisting of lung cancer (e.g.non-small cell lung cancer (NSCLC)), renal cell carcinoma (RCC),melanoma, head and neck squamous cell carcinoma (HNSCC), colorectalcancer, and ovarian cancer. It will be appreciated that a patient havinga cancer can be treated with the anti-NKG2A agent with or without aprior detection step to assess expression of HLA-E on the surface oftumor cells. Advantageously, the treatment methods can comprises a stepof detecting a HLA-E nucleic acid or polypeptide in a biological sampleof a tumor (e.g. on a tumor cell) from an individual. Example ofbiological samples include any suitable biological fluid (for exampleserum, lymph, blood), cell sample, or tissue sample. For example, atissue sample may be a sample of tumor tissue or tumor-adjacent tissue.Optionally, HLA-E polypeptide is detected on the surface of a malignantcell. A determination that a biological sample expresses HLA-E (e.g.prominently expresses; expresses HLA-E at a high level, high intensityof staining with an anti-HLA-E antibody, compared to a reference)indicates that the individual has a cancer that may have a strongbenefit from treatment with an agent that inhibits NKG2A. In oneembodiment, the method comprises determining the level of expression ofa HLA-E nucleic acid or polypeptide in a biological sample and comparingthe level to a reference level (e.g. a value, weak cell surfacestaining, etc.) corresponding to a healthy individual. A determinationthat a biological sample expresses an HLA-E nucleic acid or polypeptideat a level that is increased compared to the reference level mayindicate that the individual has a cancer that can be treated with anagent that inhibits NKG2A.

In one embodiment, a determination that a biological sample (e.g. asample comprising tumor cells, tumor tissue and/or tumor adjacenttissue) prominently expresses HLA-E nucleic acid or polypeptideindicates that the individual has a cancer that can be treated with anagent that inhibits NKG2A. “Prominently expressed”, when referring to aHLA-E polypeptide, means that the HLA-E polypeptide is expressed in asubstantial number of tumor cells taken from a given individual. Whilethe definition of the term “prominently expressed” is not bound by aprecise percentage value, in some examples a receptor said to be“prominently expressed” will be present on at least 30%, 40%, 50%, 60%,70%, 80%, or more of the tumor cells taken from a patient (in a sample).

Determining whether an individual has cancer cells that express an HLA-Epolypeptide can for example comprise obtaining a biological sample (e.g.by performing a biopsy) from the individual that comprises cancer cells,bringing said cells into contact with an antibody that binds an HLA-Epolypeptide, and detecting whether the cells express HLA-E on theirsurface. Optionally, determining whether an individual has cancer cellsthat express HLA-E comprises conducting an immunohistochemistry assay.Optionally determining whether an individual has cancer cells thatexpress HLA-E comprises conducting a flow cytometry assay.

In the treatment methods, when an anti-NKG2A antibody is administered incombination with an anti-PD-1 or anti-PD-L1 antibody, the anti-NKG2Aantibody and anti-PD-1 or anti-PD-L1 antibody can be administeredseparately, together or sequentially, or in a cocktail. In someembodiments, the anti-NKG2A is administered prior to the administrationof the anti-PD-1 or anti-PD-L1 antibodies. For example, the anti-NKG2Aantibody can be administered approximately 0 to 30 days prior to theadministration of the anti-PD-1 or anti-PD-L1 antibodies. In someembodiments, an anti-NKG2A antibody is administered from about 30minutes to about 2 weeks, from about 30 minutes to about 1 week, fromabout 1 hour to about 2 hours, from about 2 hours to about 4 hours, fromabout 4 hours to about 6 hours, from about 6 hours to about 8 hours,from about 8 hours to 1 day, or from about 1 to 5 days prior to theadministration of the anti-PD-1 or anti-PD-L1 antibodies. In someembodiments, an anti-NKG2A antibody is administered concurrently withthe administration of the anti-PD-1 or anti-PD-L1 antibodies. In someembodiments, an anti-NKG2A antibody is administered after theadministration of the anti-PD-1 or anti-PD-L1 antibodies. For example,an anti-NKG2A antibody can be administered approximately 0 to 30 daysafter the administration of the anti-PD-1 or anti-PD-L1 antibodies. Insome embodiments, an anti-NKG2A antibody is administered from about 30minutes to about 2 weeks, from about 30 minutes to about 1 week, fromabout 1 hour to about 2 hours, from about 2 hours to about 4 hours, fromabout 4 hours to about 6 hours, from about 6 hours to about 8 hours,from about 8 hours to 1 day, or from about 1 to 5 days after theadministration of the anti-PD-1 or anti-PD-L1 antibodies.

Exemplary treatment protocols for treating a human with an anti-NKG2Aantibody include, for example, administering to the patient an effectiveamount of each of an antibody that inhibits NKG2A, wherein the methodcomprises at least one administration cycle in which at least one doseof the anti-NKG2A antibody is administered at a dose of 1-10 mg/kg bodyweight. In one embodiment, the administration cycle is between 2 weeksand 8 weeks.

Exemplary treatment protocols for treating a human with an anti-NKG2Aantibody include, for example, administering to the patient an effectiveamount of each of an antibody that inhibits NKG2A and an antibody thatneutralizes the inhibitory activity of human PD-1, wherein the methodcomprises at least one administration cycle in which at least one doseof the anti-NKG2A antibody is administered at a dose of 1-10 mg/kg bodyweight and at least one dose of the anti-PD-1 or anti-PD-L1 antibody isadministered at a dose of 1-20 mg/kg body weight. In one embodiment, theadministration cycle is between 2 weeks and 8 weeks.

In one embodiment, the method comprises at least one administrationcycle, wherein the cycle is a period of eight weeks or less, wherein foreach of the at least one cycles, two, three or four doses of theanti-NKG2A antibody are administered at a dose of 1-10 mg/kg bodyweight. In one embodiment, each cycle further comprises theadministration of two, three or four doses of the anti-PD-1 oranti-PD-L1 antibody at a dose of 1-20 mg/kg body weight.

The anti-NKG2A antibody can advantageously be administered in an amountthat achieves a concentration in circulation that is at least 10, 20, or30 times higher than the concentration required for substantially full(e.g., 90%, 95%) receptor saturation (e.g., as assessed by titratinganti-NKG2A antibody on NKG2A-expressing cells, for example in PBMC), oroptionally in an amount that achieves a concentration in a extravasculartissue (e.g. the tumor tissue or environment) that is at least 10, 20,or 30 times higher than the concentration required for substantiallyfull receptor saturation (e.g., as assessed by titrating anti-NKG2Aantibody on NKG2A-expressing cells, for example in PBMC).

NKG2A+ NK cell response can be assessed using a suitable assay ofcytotoxic activity of NKG2A-expressing NK cells toward HLA-E expressingtarget cells. Examples include assays based on markers of NK cellactivation, for example CD107 or CD137 expression. The EC₅₀ for NKG2A+NK cell response (e.g., as assessed in a CD107 mobilization assay) ofblocking anti-NKG2A antibody humZ270 used in the Examples herein (e.g.having the heavy chain of any of SEQ ID NOS: 4-8 and a light chain ofSEQ ID NO: 9) is about 4 μg/ml, and the E0100 is about 10 μg/ml. Thus anamount of anti-NKG2A antibody is administered so at to maintain acontinuous (minimum) blood concentration of at least 4 μg/ml.Advantageously an amount of anti-NKG2A antibody can be administered soat to achieve and/or maintain a continuous (minimum) blood concentrationof at least 10 μg/ml. For example, the blood concentration to beachieved and/or maintained can be between 10-12 μg/ml, 10-15 μg/ml,10-20 μg/ml, 10-30 μg/ml, 10-40 μg/ml, 10-50 μg/ml, 10-70 μg/ml, 10-100μg/ml, 10-150 μg/ml or 10-200 μg/ml. When tissues outside of thevasculature are targeted (e.g. in the treatment of solid tumors), anamount of anti-NKG2A antibody is administered so at to achieve and/ormaintain a tissue concentration of at least 10 μg/ml; for example,administering an amount of anti-NKG2A antibody to achieve a bloodconcentration of at least 100 μg/ml is expected to achieve a tissueconcentration of at least 10 μg/ml. For example, the blood concentrationto be achieved and/or maintained in order to achieve/maintain 10 μg/mlin a tissue can be between 100-110 μg/ml, 100-120 μg/ml, 100-130 μg/ml,100-140 μg/ml, 100-150 μg/ml, 100-200 μg/ml, 100-250 μg/ml or 100-300μg/ml.

In some embodiments, an amount of anti-NKG2A antibody is administered soas to obtain a concentration in blood (e.g., blood serum) thatcorresponds to at least the EC₅₀ for NKG2A+ lymphocyte cell response(e.g., the NKG2A+ NK cell response), optionally at about or at leastabout, the E0100. “EC₅₀” (or “E0100”) with respect to NKG2A+ cellresponse (e.g. NK cell response), refers to the efficient concentrationof anti-NKG2A antibody which produces 50% (or 100% when referring to theEC₁₀₀) of its maximum response or effect with respect to such NKG2A+cells response (e.g. NK cell response). In some embodiments,particularly for the treatment of solid tumors, the concentrationachieved is designed to lead to a concentration in tissues (outside ofthe vasculature, e.g. in the tumor environment) that corresponds to atleast the EC₅₀ for NKG2A+ NK cell response, optionally at about, or atleast about, the E0100 for NKG2A+ NK cell response.

Exemplary treatment protocols for an anti-NKG2A antibody such as humZ270used in the Examples herein having an EC₁₀₀ for NKG2A+ NK cell responseof about 10 μg/ml comprise at least one administration cycle in which atleast one dose of the anti-NKG2A antibody is administered at a dose of2-10 mg/kg, optionally 4-10 mg/kg, optionally 6-10 mg/kg, optionally 2-6mg/kg, optionally 2-8 mg/kg, or optionally 2-4 mg/kg body weight.Optionally, at least 2, 3, 4, 5, 6, 7 or 8 doses of the anti-NKG2Aantibody are administered. In one embodiment, the administration cycleis between 2 weeks and 8 weeks. In one embodiment, the administrationcycle is 8 weeks. In one embodiment, the administration cycle is 8 weeksand comprises administering one dose of the anti-NKG2A antibody everytwo weeks (i.e. a total of four doses).

In one aspect of any of the embodiments herein, the anti-NKG2A antibodyis administered once about every two weeks.

Exemplary treatment protocols for use with an anti-NKG2A antibody,particularly for the treatment of a hematopoietic tumor, include forexample, administering to the patient an anti-NKG2A antibody two timesper month in an amount effective to maintain a continuous bloodconcentration of anti-NKG2A antibody of at least 10 μg/ml between atleast two successive administrations of the anti-NKG2A antibody isbetween 2-10 mg/kg, optionally 2-6 mg/kg, optionally 2-8 mg/kg,optionally 2-4 mg/kg, optionally 2-6 mg/kg, optionally 2-4 mg/kg,optionally about 4 mg/kg body weight. These doses can optionally beadministered so as to provide for continued blood concentration ofanti-NKG2A antibody of at least 10 μg/ml throughout the treatment cycle.Achieving blood concentration of anti-NKG2A antibody of 10 μg/mlcorresponds to the E0100 for an antibody such as humanized Z270.

Exemplary treatment protocols for use with an anti-NKG2A antibody,particularly for the treatment of a solid tumor where anti-NKG2Aantibody EC₅₀ concentration is sought in extravascular tissue (e.g., inthe tumor or tumor environment), include for example, administering tothe patient an anti-NKG2A antibody two times per month in an amounteffective to maintain a continuous blood concentration of anti-NKG2Aantibody of at least 40 μg/ml between at least two successiveadministrations of the anti-NKG2A antibody is between 2-10 mg/kg,optionally 2-6 mg/kg, optionally 2-4 mg/kg, optionally about 4 mg/kgbody weight. These doses can optionally be administered so as to providefor continued blood concentration of anti-NKG2A antibody of at least 40μg/ml throughout the treatment cycle. Achieving blood concentration ofanti-NKG2A antibody of 40 μg/ml is expected to provide a tissue (e.g.,extravascular tissue, tumor environment) concentration of about 4 μg/ml,in turn corresponding to the EC₅₀ for an antibody such as humanizedZ270.

Exemplary treatment protocols for use with an anti-NKG2A antibody,particularly for the treatment of a solid tumor where anti-NKG2Aantibody EC₅₀ concentration is sought in extravascular tissue (e.g., inthe tumor or tumor environment), include for example, administering tothe patient an effective amount of an anti-NKG2A antibody, wherein theantibody is administered 2 times per month and the amount effective tomaintain a continuous blood concentration of anti-NKG2A antibody of atleast 100 μg/ml between at least two successive administrations of theanti-NKG2A antibody is between 4-10 mg/kg, optionally 4-6 mg/kg,optionally 4-8 mg/kg, optionally about 4 mg/kg, optionally about 6mg/kg, optionally about 8 mg/kg, or optionally about 10 mg/kg. Thesedoses can optionally be administered so as to provide for continuedblood concentration of anti-NKG2A antibody of at least 100 μg/mlthroughout the treatment cycle. Achieving blood concentration ofanti-NKG2A antibody of 100 μg/ml is expected to provide a tissue (e.g.,extravascular, tumor environment) concentration of about 10 μg/ml, inturn corresponding to the E0100 for an antibody such as humanized Z270.

Further exemplary treatment protocols for use with an anti-NKG2Aantibody include regimens that employ a loading period with a higherdose, followed by a maintenance period. For example, a loading periodmay comprise administering to the patient an effective amount of ananti-NKG2A antibody, wherein the antibody is administered one or moretimes in an amount effective to maintain a continuous bloodconcentration of anti-NKG2A antibody of at least 100 μg/ml until thefirst administration of anti-NKG2A antibody in the maintenance regimen.For example, when administered once, a loading dose of 10 mg/kg ofanti-NKG2A antibody can be administered, wherein the firstadministration of anti-NKG2A antibody within the maintenance regimenoccurs about two weeks (or less) after the loading dose. The maintenanceregimen can then employ a lower dose and/or lower frequency ofadministration in order to maintain a continuous blood concentration ofanti-NKG2A antibody of at least 100 μg/ml between successiveadministrations within the maintenance regimen. For example, amaintenance regimen can comprise administering anti-NKG2A antibody everytwo weeks at a dose of between 2-10 mg/kg, optionally 4-10 mg/kg,optionally 2-4 mg/kg, optionally 4-6 mg/kg, optionally 4-8 mg/kg,optionally about 4 mg/kg, optionally about 6 mg/kg, optionally about 8mg/kg.

In certain embodiments, a dose (e.g. each dose) of the anti-NKG2Aantibody is administered at 4, 6, 8 or 10 mg/kg. In certain embodiments,a dose (e.g. each dose) of the anti-PD-1 antibody is administered at1-20 mg/kg, optionally at 10 mg/kg. In certain embodiments, a dose (e.g.each dose) of the anti-PD-L1 antibody is administered at 10, 15, 20 or25 mg/kg, optionally at 1200 mg total dose. In certain embodiments, thecombined therapy permits the anti-PD-1 or PD-L1 antibody to beadministered at a lower dose; in one embodiment, each dose of theanti-PD-1 antibody is administered at 2 or 3 mg/kg.

In one embodiment, the anti-NKG2A antibody and anti-PD-1 or anti-PD-L1antibody are administered at the following doses:

-   -   (a) 1-10 mg/kg anti-NKG2A antibody and (i) 1-10 mg/kg of        anti-PD-1 antibody or (ii) 1-20 mg/kg of anti-PD-L1 antibody;    -   (b) 4, 6, 8 or 10 mg/kg anti-NKG2A antibody and 10 mg/kg of        anti-PD-1 or anti-PD-L1 antibody;    -   (c) 4, 6, 8 or 10 mg/kg anti-NKG2A antibody and 3 mg/kg of        anti-PD-1 antibody; or    -   (d) 4, 6, 8 or 10 mg/kg anti-NKG2A antibody and 2 mg/kg of        anti-PD-1 antibody.

In one aspect of any of the embodiments herein, the anti-NKG2A antibodyis administered once about every two weeks. In one aspect of any of theembodiments herein, the anti-PD-1 or anti-PD-L1 antibody is administeredonce about every three weeks. In one aspect of any of the embodimentsherein, the anti-PD-1 or anti-PD-L1 antibody is administered once aboutevery two weeks. In one aspect of any of the embodiments herein, theanti-PD-1 or anti-PD-L1 antibody is administered once about every fourweeks.

In one embodiment the anti-PD-1 or anti-PD-L1 antibody and/or theanti-NKG2A antibody are administered by i.v. In one embodiment theanti-PD-1 or anti-PD-L1 antibody and/or the anti-NKG2A antibody areadministered on the same day, optionally further once about every twoweeks, optionally further by i.v.

In other aspects, methods are provided for identifying NKG2A+PD1+ NKcells and/or T cells. Assessing the co-expression of NKG2A and PD-1 onNK cells and/or T cells can be used in diagnostic or prognostic methods.For example, a biological sample can be obtained from an individual(e.g. from cancer or cancer-adjacent tissue obtained from a cancerpatient) and analyzed for the presence of NKG2A+PD1+ NK and/or T cells.The expression of both NKG2A and PD-1 on such cells can, for example, beused to identify individuals having tumor infiltrating NK and/or T cellswhich are inhibited by NKG2A polypeptides (and optionally further by PD1polypeptides). The method can, for example, be useful as a prognosticfor response to treatment with an agent that neutralizes NKG2A, as aprognostic for response to treatment with an agent that neutralizes PD1,or as a prognostic for response for combined treatment with an agentthat neutralizes NKG2A and an agent that neutralizes PD1.

In one embodiment, provided is a method for assessing whether anindividual is suitable for treatment with an agent that inhibits NKG2Aand an agent that neutralizes the inhibitory activity of human PD-1, themethod comprising detecting a lymphocyte population (e.g. CD8+ T cells,NK cells) that express both an NKG2A nucleic acid or polypeptide and aPD-1 nucleic acid or polypeptide in a biological sample from anindividual. A determination that the individual has a lymphocytepopulation that express both an NKG2A nucleic acid or polypeptide and aPD-1 nucleic acid or polypeptide indicates that the patient has a cancerthat can be treated with an agent that inhibits NKG2A in combinationwith an agent that neutralizes the inhibitory activity of human PD-1.

In other aspects, methods are provided for identifying NKG2A+PD1+ NKcells and/or T cells. The finding that tumor infiltrating effectorlymphocytes can express both inhibitory receptors NKG2A and PD-1 givesrise to improved treatment methods as well as methods to detect suchdouble-restricted/inhibited effector cells that can be useful indiagnostics and prognostics.

For example, a biological sample can be obtained from an individual(e.g. from cancer or cancer-adjacent tissue obtained from a cancerpatient) and analyzed for the presence of NKG2A+PD1+ NK and/or T cells.The expression of both NKG2A and PD-1 on such cells can, for example, beused to identify individuals having tumor infiltrating NK and/or T cellswhich are inhibited by both NKG2A and PD1 polypeptides. The method can,for example, be useful as a prognostic for response to treatment with anagent that neutralizes NKG2A, as a prognostic for response to treatmentwith an agent that neutralizes PD1, or as a prognostic for response forcombined treatment with an agent that neutralizes NKG2A and an agentthat neutralizes PD1.

Detecting NKG2A- and PD-1 restricted NK and/or CD8 T cells withinbiological samples can more generally have advantages for use in thestudy, evaluation, diagnosis, prognosis and/or prediction of pathologieswhere characterization of NK and/or CD8 T cells is of interest. Forexample, favorable or unfavorable cancer prognosis can be made byassessing whether tumor or tumor adjacent tissues are characterized byinfiltrating NK and/or CD8 T cells that express both NKG2A and PD-1.

For example, cancer in patients can be characterized or assessed usinganti-NKG2A and anti-PD1 antibodies to assess whether tumor-infiltratingNK and/or CD8 T cells are NKG2A+PD1+, including whether such NK and/orCD8 T cells are present at the tumor periphery (in cancer adjacenttissue). The methods can be useful to determine whether a patient has apathology characterized by NK and/or CD8 T cells which could be amenableto modulation by therapeutic agents that directly act on such NK and/orCD8 T cells (e.g. by binding to NKG2A and/or PD-1, or their respectiveligands HLA-E or PD-L1) or that indirectly act on such NK and/or CD8 Tcells (e.g., by producing cytokines or other signalling molecules thatcan modulate the activity of the NK and/or CD8 T cells). Optionally, inany embodiment, the patient has been treated with an agent thatneutralizes PD-1. The methods described herein can optionally furthercomprise administering to an individual such a therapeutic agent if itdetermined that the individual has a pathology which could be amenableto modulation by therapeutic agents that act on the tumor infiltratingNK and/or CD8 T cells.

In one aspect the inventors provides an in vitro method for detecting aNKG2A+ PD-1+ lymphocyte, optionally an NK or CD8+ T cell, the methodcomprising providing a biological sample comprising tumor infiltratinglymphocytes and determining whether the lymphocytes express NKG2A andPD-1.

In one embodiment, provided is a method for assessing whether anindividual is suitable for treatment with an agent that inhibits NKG2A(and optionally further with an agent that neutralizes the inhibitoryactivity of human PD-1), the method comprising detecting a lymphocytepopulation (e.g. CD8+ T cells) that express both an NKG2A nucleic acidor polypeptide and a PD-1 nucleic acid or polypeptide in a biologicalsample from an individual. A determination that the individual has alymphocyte population that express both an NKG2A nucleic acid orpolypeptide and a PD-1 nucleic acid or polypeptide can indicate that thepatient has a cancer that can be treated with an agent that inhibitsNKG2A in combination with an agent that neutralizes the inhibitoryactivity of human PD-1.

In other aspects, methods are provided for assessing whether anindividual having a cancer is a poor responder to treatment with anagent that neutralizes PD-1, wherein the presence and/or numbers ofNKG2A+ and/or NKG2A+PD1+ NK cells and/or T cells is assessed. Assessingthe expression of NKG2A (and/or the co-expression of NKG2A and PD-1) caninvolve for example, obtaining a biological sample from an individual(e.g. from cancer or cancer-adjacent tissue obtained from a cancerpatient) and analyzing the sample for the presence of NKG2A+ NK and/orCD8 T cells. Elevated expression of NKG2A (and optionally further PD-1)and/or numbers of such NKG2A+ and/or NKG2A+PD-1+ cells can identifyindividuals having tumor infiltrating NK and/or T cells which areinhibited by both NKG2A and PD1 polypeptides. Optionally, an increase inexpression of NKG2A (and optionally further PD-1) and numbers of suchNKG2A+ and/or NKG2A+PD-1+ cells is detected subsequent to administrationto the individual of an agent that neutralizes PD-1 (compared, e.g. to areference value or to a value before treatment with the agent thatneutralizes PD-1). An individual having elevated or increased expressionof NKG2A (and optionally further PD-1) and numbers of such NKG2A+ and/orNKG2A+PD-1+ cells can be determined to be a poor responder for an agentthat neutralize the activity of PD-1. The method can, for example, beuseful as a prognostic for response to treatment with an agent thatneutralizes NKG2A, as a prognostic for response to treatment with anagent that neutralizes PD1, or as a prognostic for response for combinedtreatment with an agent that neutralizes NKG2A and an agent thatneutralizes PD1.

In any of the methods herein, detecting NKG2A and/or PD1 on T and/or NKcells may comprise detecting NKG2A and/or PD1 expression on tissueinfiltrating human CD8 T cells and/or NK cells, said method comprisingproviding a tumor sample from an individual (e.g. a sample or tumortissue or tumor-adjacent tissue), and detecting tissue infiltrating CD8T cells and/or NK cells in said sample using a monoclonal antibody thatspecifically binds to a human NKG2A polypeptide and a monoclonalantibody that specifically binds to a human PD-1 polypeptide in thesamples. Optionally, in any embodiment, the patient has been treatedwith an agent that neutralizes PD-1. In one embodiment, the samplecomprises tumor cells, tumor tissue or tumor adjacent tissue. In oneembodiment, the CD8 T cells and/or NK cells are identified usingimmunohistochemistry methods. In one embodiment, the sample is aparaffin-embedded sample; optionally the paraffin-embedded sample hasbeen fixed, embedded in paraffin, sectioned, deparaffinized, andtransferred to a slide before being brought into contact with themonoclonal antibody. In one embodiment, the CD8 T cells and/or NK cellsare identified using flow cytometry methods.

EXAMPLES Example 1—RMA-S and A20 Tumor Cells are Infiltrated by NK CellsExpressing NKG2A and CD8 T Cells Expressing NKG2A and/or PD-1

Lymphocytes generally are not found to co-express NKG2A and PD-1. Toinvestigate the expression of these receptors on tumor-infiltratinglymphocytes, distribution of NKG2A and PD-1 were studied on NK and Tcell subsets in tumor from mice. Lymphocytes were taken from spleen,from tumor draining lymph nodes, as well as from within solid tumors.

C57/BL6 mice were engrafted (sc) with PDL-1+Qa-1+ RMA-S cells (Qa-1,Qdm, B2m) or with A20 tumor cells. RMA-S Qa-1 Qdm B2m (top row) and A20(bottom row) tumor bearing mice were sacrificed when tumor volumes werearound 500 mm³.

Results are shown in FIGS. 1A and 1B. Tumor cells (FIG. 1A) and tumorinfiltrating lymphocytes-TIL- (FIG. 1 B) were analyzed by flow cytometryrespectively for the expression of Qa-1 and PDL-1 for tumor cells andNKG2A and PD-1 for TIL. One representative mouse out of 3 is shown.MFI:Median of fluorescence intensity.

More than half of the infiltrating NK cells from both tumor typesexpressed NKG2A, suggesting that tumor-infiltrating NK cells areinhibited by NKG2A. The NKG2A+ NK cells generally did not expresssignificant amounts of PD-1. However, CD8 T cells that were positive forboth NKG2A and PD-1 were found, suggesting that the CD8 T cells may berestricted by both inhibitory receptors NKG2A and PD-1.

Example 2—NK and T Cell Subsets from Mice Bearing Rma-Rae1 Tumors areCapable of NKG2A and PD-1 Co-Expression

To further investigate the expression of receptors NKG2A and PD-1,distribution of NKG2A and PD-1 were studied on NK and T cell subsets inmice. Lymphocytes were taken from spleen, from tumor draining lymphnodes, as well as from within solid tumors.

C57/BL6 mice were engrafted (sc) with RMA-Rae clone 6 (2 million cells).These tumor cells express CD94/NKG2A ligand, Qa-1. Mice were sacrificedat day 12 with a mean tumor volume: 723 mm³, SD: 161 mm³, n=4. Followingcell suspension preparation from spleen, LN and tumor, cells werestained as follows: CD3e PerCP Cy5.5, NKP46 Alexa 647, NKG2A/C/E FITC,PD1 PE, CD8 Pacific Blue.

Results are shown in FIG. 2 and Tables 1-3.

In the NK cell subset, cells in both the draining lymph nodes and spleenwere about half NKG2A-positive and half NKG2A-negative, however inneither case was there significant expression of PD1. NK cells fromlymph nodes were NKG2A⁺ PD-1⁻ (49.2%) and NKG2A-PD-1⁻ (49.5%), and lessthan 1% (mean) of NK cells were NKG2A⁺ PD-1⁺. NK cells from spleen wereNKG2A⁺ PD-1⁻ (44.1%) and NKG2A⁻ PD-1⁻ (55.7%) and a mean of 0.1% (mean)of NK cells were NKG2A⁺ PD-1⁺.

In the T cell subset most cells were NKG2A-negative (only 1.1% in lymphnodes and 4.7% in spleen are NKG2A⁺), and a small fraction of cells werePD-1⁺ (3.5% in lymph nodes and 10% in spleen were PD-1⁺ NKG2A⁻), withoutsignificant double positive NKG2A PD-1 cells. Only 0.1% (mean) of Tcells in lymph nodes were NKG2A⁺ PD-1⁺ and only 0.4% (mean) of T cellsin spleen were NKG2A⁺ PD-1⁺. 95.1% of T cells from lymph nodes weredouble negative and 85.6% of T cells from spleen were double negative.

In the CD8 T cell subset, most cells were again NKG2A-negative (only1.6% in lymph nodes and 3.9% in spleen are NKG2A⁺), and a small fractionof cells were PD-1⁺ (1.1% in lymph nodes and 2.5% in spleen were PD-1⁺NKG2A⁻), without significant double positive NKG2A PD-1 cells. Only 0.2%(mean) of T cells in lymph nodes were NKG2A⁺ PD-1⁺ and only 0.3% (mean)of T cells in spleen were NKG2A⁺ PD-1⁺. 97.3% of T cells from lymphnodes were double negative and 93.6% of T cells from spleen were doublenegative.

However, among tumor infiltrating lymphocytes (TIL), all cells subsetshad cells expressing PD-1. NK cells, which were not previously found insignificant percentages to express PD-1, were observed in tumor to bePD-1-positive, including within the NKG2A⁺ subset, with 31.8% (mean) ofNK cells that were NKG2A⁺ PD-1⁺. While almost no CD8 T cells outside thetumor had NKG2A expression, CD8 T cells expressing PD-1 were frequent inthe tumor (the tumor infiltrating CD8 T cell subset had a mean of 26.3%NKG2A+ positive cells). Moreover, within this NKG2A-positive CD8 T cellsubset, most of the cells were NKG2A⁺ PD-1⁺ (19.4% (mean). Yet, amongthe CD8⁻ T cell subset, there was little difference in NKG2A expressionobserved between TILs and spleen or lymph node cells, as only 5.1% of Tcells in the tumor expressed NKG2A, and only 3.6% of T cells were doublepositive NKG2A PD-1.

TABLE 1 Spleen % among NK % NK % NK % NK % NK NKG2A− NKG2A+ NKG2A+NKG2A− % NK PD1+ PD1+ PD1− PD1− NKG2A+ Mice1 0.064 0.064 41.8 58.141.864 Spleen Mice2 0.15 0.098 46.1 53.7 46.198 Spleen Mice4 0.19 0.1944.3 55.3 44.49 Spleen Mean 0.1 0.1 44.1 55.7 44.2 SD 0.1 0.1 2.2 2.22.2 % among T lymphocytes % T % T % T % T NKG2A− NKG2A+ NKG2A+ NKG2A− %T PD1+ PD1+ PD1− PD1− NKG2A+ Mice1 8.08 0.38 4.52 87 4.54 Spleen Mice29.64 0.32 4.22 85.8 6.25 Spleen Mice4 12.3 0.41 3.21 84.1 3.37 SpleenMean 10.0 0.4 4.0 85.6 4.7 SD 2.13 0.05 0.69 1.46 1.45 % among CD8+ Tlymphocytes % TCD8+ % TCD8+ % TCD8+ % TCD8+ NKG2A− NKG2A+ NKG2A+ NKG2A−% T CD8+ PD1+ PD1+ PD1− PD1− NKG2A+ Mice1 1.8 0.27 4.68 93.2 4.95 SpleenMice2 2.08 0.27 3.09 94.6 3.36 Spleen Mice4 3.67 0.48 2.97 92.9 3.45Spleen Mean 2.5 0.3 3.6 93.6 3.9 SD 1.01 0.12 0.95 0.91 0.89

TABLE 2 Tumor Draining Lymph Nodes % among NK % NK % NK % NK % NK NKG2A−NKG2A+ NKG2A+ NKG2A− % NK PD1+ PD1+ PD1− PD1− NKG2A+ Mice1 LN 0.68 0.8545.10 53.40 45.95 Mice3 LN 0.20 0.40 54.70 44.70 55.10 Mice4 LN 0.611.21 47.90 50.30 49.11 Mean 0.50 0.82 49.23 49.47 50.05 SD 0.26 0.414.94 4.41 4.65 % among T lymphocytes % T % T % T % T NKG2A− NKG2A+NKG2A+ NKG2A− % T PD1+ PD1+ PD1− PD1− NKG2A+ Mice1 LN 2.8 0.1 1.8 95.30.5 Mice3 LN 6.6 0.4 2.4 90.6 0.7 Mice4 LN 2.1 0.0 0.6 97.2 0.6 Mean 3.50.1 1.3 95.1 1.1 SD 2.1 0.2 0.9 3.1 1.1 % among CD8+ T lymphocytes %TCD8+ % TCD8+ % TCD8+ % TCD8+ NKG2A− NKG2A+ NKG2A+ NKG2A− % T CD8+ PD1+PD1+ PD1− PD1− NKG2A+ Mice1 LN 1.0 0.2 2.0 96.8 2.1 Mice3 LN 2.1 0.6 2.594.8 3.1 Mice4 LN 0.6 0.1 0.7 98.6 0.8 Mean 1.1 0.2 1.4 97.3 1.6 SD 0.70.3 1.0 1.9 1.3

TABLE 3 Tumor Infiltrating Lymphocytes % among NK % NK % NK % NK % NKNKG2A− NKG2A+ NKG2A+ NKG2A− % NK PD1+ PD1+ PD1− PD1− NKG2A+ Mice1 TIL18.8 25.6 26.6 28.9 52.2 Mice2 TIL 22.8 31 13.5 32.7 44.5 Mice3 TIL 2638.2 13.8 22 52 Mice4 TIL 24.4 32.5 22.5 20.7 55 Mean 23 31.8 19.1 26.150.9 SD 3.1 5.2 6.5 5.7 4.5 % among T lymphocytes % T % T % T % T NKG2A−NKG2A+ NKG2A+ NKG2A− % T PD1+ PD1+ PD1− PD1− NKG2A+ Mice1 TIL 51.7 4.857.8 35.6 2.89 Mice2 TIL 86.5 1.47 1.42 10.7 8.22 Mice3 TIL 75 4.93 3.2916.8 9.3 Mice4 TIL 66 3.1 6.2 24.7 0 Mean 69.8 3.6 4.7 22.0 5.1 SD 14.71.6 2.9 10.8 4.4 % among CD8+ T lymphocytes % TCD8+ % TCD8+ % TCD8+ %TCD8+ NKG2A− NKG2A+ NKG2A+ NKG2A− % T CD8+ PD1+ PD1+ PD1− PD1− NKG2A+Mice1 TIL 49.5 16 10.8 23.7 26.8 Mice2 TIL 52.2 21.7 4.35 21.7 26.05Mice3 TIL 44.3 28.6 5.71 21.4 34.31 Mice4 TIL 52.8 11.3 6.6 29.2 17.9Mean 49.7 19.4 6.9 24.0 26.3 SD 3.9 7.5 2.8 3.6 6.7

Example 3—NKG2A and PD-1 Expression in Tumor Bearing Mice

To further investigate NKG2A and PD-1 expression in tumor-bearing mice,C57/BL6 mice were engrafted (sc) with different tumor cells, eitherRMA-Rae1, MC38 or RMA lines. To evaluate the influence of tumor volume,mice were sacrificed when their tumors reached respectively the volumesof 500, 2000 and 800 mm³.

Results are shown in FIGS. 3A and 3B, with RMA Rae1 (top row), MC38(medium row) and RMA (bottom row). NK cells (FIG. 3A) and CD8 T cells(FIG. 3B) were analyzed by flow cytometry in spleen, tumor draininglymph node (LN) and tumor for NKG2A and PD-1 expression. Onerepresentative mouse out of 2 to 4 is shown.

In the NK cell subset, cells in the tumor, lymph nodes and spleen wereabout half NKG2A-positive and half NKG2A-negative. Neither NK cells(regardless of their NKG2A expression) from the draining lymph nodes northe spleen showed any significant expression of PD1. However, the tumorinfiltrating NK cells from RMA-Rae1 and RMA expressed significant levelsof both NKG2A and PD1. Tumor infiltrating NK cells from tumor line MC38that were sacrificed with particularly large volume (2000 mm³) expressedNKG2A (50%) but did not significantly express PD1 (3%).

Unlike NK cells which express NKG2A in about half the population, theCD8 T cells from spleen and lymph nodes generally expressed neitherNKG2A nor PD1. However, in tumors, a large proportion of CD8 T cellsexpressed both NKG2A and PD1 (28% in RMA-Rae1, 43% of MC38 and 40% ofRMA were double positive). The results again suggest that tumorinfiltrating CD8 T cells as well as NK cells may be capable of beingrestricted by both inhibitory receptor NKG2A and PD1, furthermore acrossdifferent types of tumor cells.

Example 4—Increased NKG2A Expressing Tumor Infiltrating CD8 T Cells inMice Resistant to PD-1 Treatment

To evaluate the effect of treatment with anti-PD1 antibody on CD8 Tcells, MC38 tumor bearing mice were either treated with 200 μg of ratIgG2a isotype control (IC) or neutralizing anti-mouse PD-1 monoclonalantibodies on days 11, 14 and 17 after cells engraftment. Mice(n=3/group) were sacrificed on day 31 and CD8 T cells were characterizedby flow cytometry in spleen, tumor draining lymph node (LN) and tumor.Means+/−SD (n=3) of the percentages of CD8 NKG2A+ among CD8 T cells arerepresented. P<0.005 (***), P<0.0005 (****), statistical analysisperformed with Two way ANOVA followed by Tukey's multiple comparisontest.

Results are shown in FIG. 4 . Similarly to that observed in otherexperiments, CD8 T cells from spleen of lymph nodes did notsignificantly express NKG2A. Administration of anti-PD1 antibody did notcause any change in the level of NKG2A expression in the spleen or lymphnode T cells. However, in the tumor infiltrating CD8 T cell population,administration of anti-PD1 antibody caused a more than 50% increase inNKG2A-expressing CD8 T cells. The results suggest that in non respondingmice there is a significant increase of CD8 T cells expressing NKG2A incomparison to mice treated with isotypic control mAb. NKG2A receptor maytherefore have an increased contribution to the inhibition of the CD8 Tcell response toward tumors in poor responders to anti-PD1.Neutralization of NKG2A may therefore be useful to reverse theinhibition of the NKG2A restricted T cells in individuals treated withan agent that neutralizes the PD-1 axis such as an anti-PD1 or PDL1antibody.

Example 5—Combinatorial Anti-NKG2A/Anti-PD1 Blockade Inhibits TumorGrowth

To evaluate the effect of combination treatment with neutralizinganti-PD1 antibody and neutralizing anti-NKG2A antibody, C57BL/6 micewere engrafted (sc) with RMA-S Qa-1 Qdm B2m tumor cells and treated withneutralizing anti-PD1 agent (a neutralizing anti-PD-L1 antibody) andneutralizing anti-NKG2A antibody.

Briefly, C57BL/6 mice were randomized on day 11 when RMA-S Qa-1 Qdm B2mtumor volume were about 85 mm³ (n=8 mice/group) and treated with isotypecontrol, anti-mouse NKG2A mAb (200 μg, iv), anti-mouse PD-L1 mAb (200μg, ip) or anti-mNKG2A/mPDL-1 combination on days 11, 14 and 18. Tumorvolume was measured twice a week; mice were sacrificed when tumorsbecame large (volume>2000 mm³), ulcerated or necrotic, and NK and CD8 Tcells were characterized by flow cytometry.

The evolution of median tumor volume over time is shown in FIG. 5 .While anti-NKG2A yielded only a modest anti-tumor effect compared toisotype control in this model and anti-PD-L1 yielded a substantialanti-tumor effect but with tumor volume increasing toward day 28, thecombined treatment with anti-NKG2A and anti-PD-L1 completely abolishedtumor growth, with no significant growth in tumor volume observed at day28.

Example 6—an In Vivo Model of PD-1/-L1 Resistant Cancer

The A20 cell line is a mouse B cell lymphoma line that expresses PD-L1,but not Qa-1^(b). Upon subcutaneous injection into Balb/c mice, A20formed solid tumors in which PD-L1 expression was retained and whereQa-1^(b) expression was induced. A20 tumor growth was controlled by NKand CD8 T cells.

To evaluate the effect of combination treatment with neutralizinganti-PD1 antibody or neutralizing anti-PDL1 antibody, the Balb/c mice(female, 8-10 weeks old) were engrafted (sc) with A20 tumor cells (1×10⁶cells) and treated with isotype control (IC) or neutralizing anti-murinePD-1 antibody or neutralizing anti-murine PD-L1 antibody (200 μg ofanti-PD1 or anti-PD-L1, ip, days 13, 17 and 20). Tumor volume wasmeasured twice a week; mice were sacrificed when tumors became large(volume>2000 mm³), ulcerated or necrotic, and NK and CD8 T cells werecharacterized by flow cytometry.

The evolution of individual tumor volumes over time is shown in FIG. 6 .Neither anti-PD-L1 nor anti-PD-1 antibodies yielded a substantialanti-tumor effect. Among 9 mice treated with isotype control (IC), nopartial regressions, temporary complete regressions or completeregressions were observed. Among 9 mice treated with anti-PD-1 antibodytreatment, no mice had partial regressions or temporary completeregressions were observed, and 2 mice had complete regressions. Among 10mice treated with anti-PD-L1 antibody, no partial regressions ortemporary complete regressions were observed, and 2 mice experiencedcomplete regressions.

In spite of high expression of PD-1 on many immune infiltrating cells,and high expression of PD-L1 on tumor cells, monotherapy with anti-PD-1or -PD-L1 mAb resulted in only moderate reduction in tumor growth.Interestingly, more than 50% of A20 tumor infiltrating NK cells andabout 10% of CD8 T cells expressed CD94/NKG2A. The NKG2A+CD8 T cellpopulation also co-expressed PD-1. Qa-1b expression was induced not onlyon the surface of tumor cells but also on infiltrating immune cells invivo.

Example 7—Anti-NKG2A Antibody and Anti-PD1 Antibody Lead to CompleteTumor Regression in PD-1/-L1 Resistant A20 Tumors

As the A20 tumor cells co-expressed both PD-L1 and Qa-1^(b), micebearing A20 tumors and receiving neutralizing anti-PD-1 antibody (seeExample 6) were then additionally treated with neutralizing anti-NKG2Aantibody.

Balb/c mice (female, 8-10 weeks old) were engrafted (sc) with A20 tumorcells (1×10⁶ cells) and treated with isotype control (IC), neutralizinganti-murine PD-1 antibody, or anti-PD-1 and neutralizing anti-NKG2Aantibody (200 μg, ip, days 10, 13 and 17). Tumor volume was measuredtwice a week with a caliper. Animals were euthanized when tumor becamelarge (volume>2000 mm³), ulcerated or necrotic. Data representindividual tumor volumes per experiment.

The evolution of individual tumor volumes over time is shown in FIG. 7 .Similarly to that observed in Example 6, tumors are resistant toanti-PD-1 antibodies or isotype control (IC). Among 10 mice treated withisotype control (IC), no partial regressions, one temporary completeregression and one complete regression were observed, while withanti-NKG2A alone one partial regression, no temporary completeregressions and one complete regression were observed. Among 9 micetreated with anti-PD-1 antibody treatment, no mice had partialregressions, 1 temporary complete regression was observed, and 3complete regressions were observed. However, when the anti-NKG2Aantibody is added (labelled Combination in FIG. 7 ), among the 10 micetreated with the addition of anti-NKG2A antibody, 7 complete regressionswere observed (no partial regressions or temporary completeregressions). Anti-NKG2A antibody therefore has a promising therapeuticpotential in the treatment of cancers that are resistant to treatmentwith anti-PD-1 or anti-PD-L1 antibodies.

Example 8—Anti-NKG2A Antibody and Anti-PD-L1 Antibody Lead to CompleteTumor Regression in the Anti-PD1/-L1-Resistant A20 Tumors

Balb/c mice (female, 8 weeks old, n=11/group) were engraftedsub-cutaneously with A20 tumor cells (5×10⁶ cells) and mice wererandomized on day 11 (tumor volume around 50 mm³) and treated withisotype controls (IC), neutralizing anti-PD-L1 antibody (50 μg, ip, days11, 14, 18, 21, 25, 28), blocking anti-NKG2A mAb (200 μg, iv, days 11,14 and 18) or the combination of both mAbs. Tumor volume was measuredtwice a week with a caliper. Animals were euthanized when tumor volumewas above 2000 mm³, ulcerated or necrotic. Data represent individualtumor curve. PR:Partial Regression, CR:Complete Regression

The evolution of individual tumor volumes over time is shown in FIG. 8 .Similarly to that observed in Example 6, tumors are resistant toanti-PD-L1 antibodies or isotype control (IC). Among 11 mice treatedwith isotype control (IC), no partial regressions and two completeregressions (18%) were observed, while with anti-NKG2A alone fourcomplete regressions were observed. Among 11 mice treated withanti-PD-L1 antibody treatment alone, no mice had partial regressions and6 complete regressions were observed. However, when the anti-NKG2Aantibody is added, among the 11 mice treated with the addition ofanti-NKG2A antibody, 9 complete regressions were observed. The resultsillustrate that anti-NKG2A antibody therefore has a promisingtherapeutic potential in the treatment of cancers that are resistant totreatment with anti-PD-L1 antibodies.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Where“about” is used in connection with a number, this can be specified asincluding values corresponding to +/−10% of the specified number.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having,” “including,” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

1. (canceled)
 2. A method of treating a cancer in an individualcomprising administering an antibody that neutralizes the activity ofNKG2A in combination with an antibody that neutralizes the activity ofPD-1, wherein: a) the antibody that neutralizes the activity of NKG2Acomprises heavy chain CDR1, CDR2 and CDR3 domains of a heavy chainhaving the sequence set forth in any one of SEQ ID NOs: 4, 5, 6, 7, or 8and CDR1, CDR2 and CDR3 domains of a light chain having the sequence ofSEQ ID NO: 9; b) the antibody that neutralizes the activity of PD-1 isselected from the group consisting of pembrolizumab, nivolumab, AMP-514,MEDI-4736, pidlizumab and MPDL3280A; and c) the cancer is: i) selectedfrom the group consisting of squamous cell cancer, bladder cancer,breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer,ovarian cancer, head and neck cancer, prostate cancer, pancreaticcancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer;leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia,acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, Burketts lymphoma,multiple myeloma; acute myelogenous leukemia, chronic myelogenousleukemia, promyelocytic leukemia, myelodysplastic syndrome,fibrosarcoma, rhabdomyosarcoma; melanoma, seminoma, terato-carcinoma,neuroblastoma, glioma; astrocytoma, neuroblastoma, glioma, schwannomas,and thyroid follicular cancer; ii) selected from the group consisting ofa non-small cell lung cancer (NSCLC), kidney cancer, gastrointestinalcancer, pancreatic adenocarcinoma, esophagus adenocarcinoma, breastcancer, renal cell carcinoma (RCC), melanoma, colorectal cancer andovarian cancer; or iii) an advanced and/or refractory solid tumor; oriv) an advanced refractory solid or hematological tumor or a progressingsolid or hematological tumor.
 3. The method according to claim 2,wherein the cancer is a head and neck cancer.
 4. The method according toclaim 3, wherein the head and neck cancer is head and neck squamous cellcarcinoma (HNSCC).
 5. The method according to claim 2, wherein thecancer is a lung cancer.
 6. The method according to claim 5, wherein thelung cancer is a non-small cell lung cancer (NSCLC).
 7. The methodaccording to claim 2, wherein the cancer is a non-small cell lung cancer(NSCLC), kidney cancer, gastrointestinal cancer, pancreaticadenocarcinoma, esophagus adenocarcinoma, breast cancer, renal cellcarcinoma (RCC), melanoma, colorectal cancer or ovarian cancer.
 8. Themethod according to claim 2, wherein the antibody that neutralizes theactivity of NKG2A comprises a heavy chain comprising SEQ ID NO: 4 and alight chain comprising SEQ ID NO:
 9. 9. The method according to claim 2,wherein the antibody that neutralizes the activity of NKG2A comprises aheavy chain comprising SEQ ID NO: 5 and a light chain comprising SEQ IDNO:
 9. 10. The method according to claim 2, wherein the antibody thatneutralizes the activity of NKG2A comprises a heavy chain comprising SEQID NO: 6 and a light chain comprising SEQ ID NO:
 9. 11. The methodaccording to claim 2, wherein the antibody that neutralizes the activityof NKG2A comprises a heavy chain comprising SEQ ID NO: 7 and a lightchain comprising SEQ ID NO:
 9. 12. The method according to claim 2,wherein the antibody that neutralizes the activity of NKG2A comprises aheavy chain comprising SEQ ID NO: 8 and a light chain comprising SEQ IDNO:
 9. 13. The method according to claim 2, wherein the antibody thatneutralizes the activity of PD-1 is MEDI-4736.
 14. The method accordingto claim 2, wherein the antibodies are formulated for combinedadministration.
 15. The method according to claim 2, wherein theantibodies are formulated for separate administration and areadministered concurrently or sequentially.
 16. The method according toclaim 2, wherein said cancer is an advanced and/or refractory solidtumor.
 17. The method according to claim 2, wherein said cancer is anadvanced refractory solid or hematological tumor or a progressing solidor hematological tumor.